Are NETs involved in fighting Leptospira interrogans infections?

Neutrophils are the most abundant white blood cells in the bloodstream.  As the first immune cells to be recruited to infected tissues, they play a key role in the fighting microbial intruders.  It's long been known that they engulf microbes by phagocytosis, which results in the microbes being imprisoned within phagosomes inside the neutrophil.  Deadly proteases, antimicrobial proteins, and reactive oxygen species are released into the phagosome to kill the microbes.

Another means used by neutrophils to kill microbes was discovered just a decade ago.  When mixed with bacteria, neutrophils cast nets of DNA impregnated with antimicrobial proteins to trap and kill the bacteria.  The web-like DNA goes by the name "neutrophil extracellular trap" (NET).  Several bacteria are known to trigger neutrophils to cast NETs, and NETs have even been observed by microscopy within infected tissues.

Fluorescence staining of a neutrophil exudate in an appendicitis case.  NETs are the fibrous material.  Figure 4H from Brinkmann et al., 2004.  Bar = 50 μm.

A study published last year in PLOS NTD showed that the spirochete Leptospira interrogans is also killed by NETs.  The image below shows the spirochetes trapped in a NET cast by a human neutrophil.

Human neutrophils were cultured with L. interrogans for 3 hours.  Figure 1A from Scharrig et al., 2015.  Bar = 50 μm.

The real question is whether NETs are involved in killing L. interrogans during infection.  To answer this question, the investigators turned to the mouse model of leptospirosis.  They found that the number of spirochetes in the bloodstream more than doubled when the neutrophils in the mice were depleted by injection of a monoclonal antibody targeting a antigen located on the neutrophil surface.  Later in the infection, there was 10-fold more spirochetes in the kidneys of mice whose neutrophils were depleted than in those with normal numbers of neutrophils.  This confirmed that neutrophils were involved in limiting infections by L. interrogans, but did the neutrophils fight the infection by casting NETs?

The investigators used an indirect method to measure the amount of NETs generated during infection.  Neutrophils often expel nuclear DNA in the form of nucleosomes to generate NETs.  (Nucleosomes are assembled by wrapping nuclear DNA around histones.)  For this reason, the investigators measured the levels of free nucleosomes in the bloodstream of infected mice by ELISA. They concluded that NETs were generated by neutrophils in the bloodstream because they detected free nucleosomes in blood drawn from infected mice.  Much less was detected when neutrophils were first depleted with the anti-neutrophil antibody, confirming that the main source of free nucleosomes was neutrophils.

These results don't convince me that NETs are generated by neutrophils during L. interrogans infection.  There could be other reasons for free nucleosomes being present in the bloodstream.  For example, nucleosomes could be released from neutrophils simply dying from their battle against L. interrogans.  More convincing evidence would be direct observation of NETs in infected animals, as done in this study of mice with E. coli blood infections.

References

Scharrig E, Carestia A, Ferrer MF, Cédola M, Pretre G, Drut R, Picardeau M, Schattner M, & Gómez RM (2015). Neutrophil extracellular traps are involved in the innate immune response to infection with Leptospira. PLoS Neglected Tropical Diseases, 9 (7) PMID: 26161745

Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, & Zychlinsky A (2004). Neutrophil extracellular traps kill bacteria. Science (New York, N.Y.), 303 (5663), 1532-5 PMID: 15001782

The genomes of 20 species of Leptospira

A massive study describing the genomes of 20 species of Leptospira was published a few weeks ago in PLOS Neglected Tropical Diseases.  The deluge of sequence information will be valuable to those in the leptospirosis field.  Scientists will be able to examine differences in genetic content between various categories of Leptospira species to generate hypotheses for experimental testing.  For example, genes present in species that cause infections but missing in species that don't may be important factors responsible for the pathogenesis of Leptospira.  The genome information will also aid in vaccine and serodiagnostics development by allowing researchers to identify protein antigens that are conserved among Leptospira species circulating within a region of interest.

The 20 Leptospira species are divided into 14 infectious and six noninfectous species.  (Actually, there are now 22 species known but only 20 when this study was launched.)  The infectious species are divided further into nine pathogenic and five "intermediate" species based on their genetic relatedness.

The Venn diagram below shows the number of genes that are shared among and within the three categories of Leptospira and Leptonema illini, a closely-related spirochete.  Looking at the relevant intersection (overlap) in the diagram, there are 255 genes that are carried by infectious Leptospira but not by saprophytic Leptospira.  (The other two figures in the overlap are the number of shared genes tabulated using looser criteria.  In these cases there are 302 genes found in all but one infectious Leptospira and 369 genes when those found in the majority of infectious species are counted.)  Similarly, there are 109 genes unique to the pathogenic species (or 161 or 416, if you want to use less stringent criteria).  The small circles at the periphery show the number of genes unique to each species.  So for example, L. interrogans, the species favored for study in molecular biology labs, has 672 genes that are not found in any other Leptospira species.

Figure 2A from Fouts et al., 2016.  Source.

Reference

Fouts DE, Matthias MA, Adhikarla H, Adler B, Amorim-Santos L, Berg DE, Bulach D, Buschiazzo A, Chang YF, Galloway RL, Haake DA, Haft DH, Hartskeerl R, Ko AI, Levett PN, Matsunaga J, Mechaly AE, Monk JM, Nascimento AL, Nelson KE, Palsson B, Peacock SJ, Picardeau M, Ricaldi JN, Thaipandungpanit J, Wunder EA Jr, Yang XF, Zhang JJ, & Vinetz JM (2016). What makes a bacterial species pathogenic?: Comparative genomic analysis of the genus Leptospira. PLoS Neglected Tropical Diseases, 10 (2) PMID: 26890609

Lighting up Leptospira interrogans to test antibiotic treatment of chronically-infected mice

A group at the Pasteur Institute succeeded in generating a bioluminescent strain of Leptospira interrogans.  Their study was published last year in PLOS Neglected Tropical Diseases.

The benefit of having a bioluminescent strain is that infections of small laboratory rodents can be monitored without sacrificing the animals.  Instead, the animals are placed in a special whole-body imager that detects light emitted from the bodies.  The location of the infection in the animals can be determined from the images, and the light intensity measured by the imager gives an idea of the bacterial load in infected tissues.  After imaging, the animal can be returned to its cage, and additional images of the same animal can be taken later as the infection progresses.

Another advantage of using bioluminescent bacteria is that the luciferase reaction requires ATP, meaning that the bacteria must be metabolically active to light up.  Dead bacteria containing luciferase will not generate light, unlike green fluorescent protein (GFP), another reporter used to image bacteria (see this post that describes an experiment with Borrelia burgdorferi expressing GFP).

To generate bioluminescent L. interrogans, the researchers hooked the firefly luciferase gene up to a strong L. interrogans promoter and inserted the construct into a transposon carried on a suicide plasmid.  The plasmid was then introduced into L. interrogans by conjugation (see this blog post for details of the process).  Cultures of the engineered spirochetes lit up when luciferin, the luciferase substrate, was added.  The amount of light emitted depended on the culture density: more light was detected at higher culture densities.

Mice are ideal models to study persistent infection of the kidney since many rodents are chronic carriers of Leptospira out in nature.  These rodents don't get sick from the infection, but they contaminate the environment with the spirochete every time they urinate.

To see how chronic infection is established, the investigators injected a sublethal dose of 10⁷ bioluminescent L. interrogans cells into the abdominal cavity of C57BL6/J mice and took sequential images of the animals over the following months.  Albino mice were used because dark fur blocks the signal emitted by the bioluminescent bacteria.  (They later showed that standard C57BL6/J mice with black fur could be used as long as they shaved the fur off before placing them in the imager.)  The mice were injected with luciferin 10 minutes prior to imaging.

There turned out to be two phases of infection (see the "MFlum1" plot in the graph below).  In the acute phase, the bioluminescent signal rose to a peak by day 4 and quickly declined to background levels by day 7.  The signal then started increasing again slowly and plateaued after a month.

Figure 2A from Ratet et al., 2014.  Images of a single mouse taken sequentially are shown below the graph.  Click for larger image.  Source.

Images of a single mouse taken at different times after inoculation are shown below the graph.  Thirty minutes after inoculation, signal was detected in the abdominal cavity.  By day 3, the signal consumed the entire mouse.  At this point, the bacteria were probably circulating in the bloodstream.  By day 6, the signal was almost completely gone.  After day 6, the signal appeared again, but it was confined to the kidneys.  The intensity of the signal in the kidneys increased with time.  They did not detect signal anywhere else in the animals during the second phase.  They even sacrificed some of the infected mice 2 months into the infection to check the organs directly, but they failed to detect Leptospira by bioluminescence and qPCR in the brain, lungs, spleen, liver, or blood.  Not surprisingly, bioluminescence was detected in urine, confirming that the mice were shedding live L. interrogans.

Next, the investigators tested the effectiveness of antibiotics in treating mice infected with the bioluminescent L. interrogans.  Several antibiotics are used to treat acute leptospirosis in humans, including penicillin and azithromycin.  It is generally believed that antibiotics are more effective if provided early in acute illness.  Therefore, the investigators tested whether the timing of antibiotic treatment was important for effectiveness.

As expected, penicillin treatment was most effective when treatment was started at the beginning of the acute phase.  In mice treated with daily injections of penicillin for 5 days starting a day after infection, no bioluminescence was detected in the kidneys, and urine was free of L. interrogans as measured by qPCR.  However, if treatment was delayed until three days after inoculation, during the peak of acute infection, a low level of L. interrogans was detected in urine by qPCR even though no bioluminescence was detected in the kidneys.  It is likely L. interrogans was present in the kidneys but at levels too low to be detected by the imager. The bioluminescence approach clearly does not have the sensitivity of qPCR.  Additional experiments revealed that the limit of detection was 100 bioluminescent L. interrogans cells in 100 μl of buffer.

Penicillin was even less effective when administered after the spirochetes settled in the kidneys. When penicillin treatment was initiated at peak bacterial load in the kidneys, day 25 of infection, the signal diminished by over 90% but then bounced back to the level observed before treatment began (see figure below).  Ciprofloxacin also failed to eradicate the bacteria.

Figure 5A from Ratet et al., 2014.  Antibiotics were administered for 5 days started on day 25 of infection.  Cipr, ciprofloxacin; Pen, penicillin.  Source.

On the other hand, azithromycin managed to knock the signal in the kidneys down to background levels (see graph below).  However, the signal came back within a week, although not to the high levels seen in untreated mice.  A second course of antibiotics starting on day 112 knocked the signal back down to near background levels, but again, spirochete numbers rebounded, although not to the levels seen before retreatment.

Figure 5B from Ratet et al., 2014.  Azithromycin was administered for 5 days starting on day 25 and day 112 of infection.  Source.

Why are antibiotics ineffective in eradicating L. interrogans during the chronic phase?  Like other bacteria, L. interrogans can form biofilms in vitro.  Scientists who work with Leptospira believe that they also assemble into biofilms within the kidney tubules during chronic infection.  Biofilms are hard to eliminate in part because they harbor persister cells that tolerate antibiotics.  (See this post for some background on persister cells.)

I should caution readers from concluding that tolerance accounts for the poor effectiveness of antibiotics in treating human cases of acute leptospirosis.  As the authors point out, leptospirosis patients die because the infection severely injure vital organs.  By the time lethal damage occurs, it does not matter whether antibiotics kill all of the spirochetes.

So does the mouse model have any relevance to human leptospirosis?  The authors argue that asymptomatic carriage of Leptospira has been overlooked.  A 2013 study from the Netherlands revealed that 21% of patients who contracted  leptospirosis continued to suffer from headaches, muscle aches, and extreme fatigue two years later.  This may reflect unrepaired tissue damage inflicted during acute infection, but no one checked for the presence of Leptospira in these patients.  Another study from Peru (see this post) describes asymptomatic individuals who may have persistent Leptospira infection. Kidney function was not checked in the Peruvians, but there is reason to believe that chronic infection affects the kidneys despite the lack of symptoms.  Mice chronically infected with L. interrogans are not visibly sick, but they end up with scarred kidneys (fibrosis), as explained in this study.

If persistent asymptomatic infections really do occur in humans, it may be sensible to treat with antibiotics.  The chronically-infected mouse will serve as a nice model for testing antibacterial regimens that target Leptospira living in the kidneys.

References

Ratet G, Veyrier FJ, Fanton d'Andon M, Kammerscheit X, Nicola MA, Picardeau M, Boneca IG, & Werts C (2014). Live imaging of bioluminescent Leptospira interrogans in mice reveals renal colonization as a stealth escape from the blood defenses and antibiotics. PLoS Neglected Tropical Diseases, 8 (12) PMID: 25474719

Goris MG, Kikken V, Straetemans M, Alba S, Goeijenbier M, van Gorp EC, Boer KR, Wagenaar JF, & Hartskeerl RA (2013). Towards the burden of human leptospirosis: duration of acute illness and occurrence of post-leptospirosis symptoms of patients in the Netherlands. PloS One, 8 (10) PMID: 24098528

Fanton d'Andon M, Quellard N, Fernandez B, Ratet G, Lacroix-Lamandé S, Vandewalle A, Boneca IG, Goujon JM, & Werts C (2014). Leptospira Interrogans induces fibrosis in the mouse kidney through Inos-dependent, TLR- and NLR-independent signaling pathways. PLoS Neglected Tropical Diseases, 8 (1) PMID: 24498450


Related posts

• Life after leptospirosis, a pilot study
• Healthy human carriers of the spirochete Leptospira in the Peruvian Amazon
• The magic of antibiotic tolerance

"...and a dog with lepto in its pee."

I saw this video over at the Worms & Germs blog.  It's a new take on a popular Christmas carol.  Enjoy!

Lyrics
On the twelfth day of Christmas my true love gave to me,
Twelve tubs of Purell,
Eleven raccoon roundworms,
Ten cats-a-scratching
Nine hungry hookworms,
Eight dogs-a-biting,
Seven cats with ringworm,
Six big fat dog ticks,
Five cats with fleas.
Four rats with cowpox.
Three tapeworms,
Two toxic turtles,
And a dog with lepto in its pee.

Towards sterilizing immunity against Leptospira with a DNA vaccine

In my previous post, I described the failure of researchers to come up with a conventional protein-based subunit vaccine that confers sterilizing immunity against leptospirosis.  What I mean by "conventional" subunit vaccine is a mixture of purified recombinant Leptospira protein with an adjuvant (either aluminum hydroxide or Freund's).  Although an antibody response was detected against most proteins tested, immunization failed to prevent kidney colonization in every case, including those animals that survived challenge with lethal strains of Leptosipra.  It's becoming clear that the leptospirosis vaccine field must move beyond simple formulations of protein plus adjuvant if sterilizing immunity is desired.

Several labs have explored more modern approaches to delivering leptospirosis vaccines.  Odir Dellagostin's group down in Brazil tested the efficacy of DNA vaccines in protecting hamsters against leptospirosis, as described in this paper in Clinical and Vaccine Immunology.  Forster and colleagues targeted the Leptospira interrogans LigA and LigB proteins, which are surface proteins that disrupt (or exploit) multiple host functions.

The investigators cloned various fragments of the lengthy ligA and ligB genes downstream of the human cytomegalovirus promoter of the commercial expression plasmid pTARGET.  They mixed the plasmid DNA with aluminum hydroxide adjuvant and injected the material into the muscle of the hind leg of hamsters.  The animals were given a booster with the same material 21 days later.  An IgG immune response was detected against four of the the five Lig protein fragments being tested (see figure below).

Figure 2 from Forster et al., 2013.  Sera were drawn before immunization and after the first and second immunizations with pTARGET-based lig plasmid DNA.  Purified recombinant protein encoded by each plasmid was used as antigen in ELISAs.  Left, middle, and right bar for each DNA: before immunization, after first immunization, and after second immunization, respectively.

21 days after the boost, the animals were challenged with a lethal strain of L. interrogans.  The survival curves are shown below.  Note that animals immunized with the vector alone (small filled circles) were all dead by day 11.

Figure 3 from Forster et al., 2013.

Among the lig gene fragments, the one expressing the "LigBrep" fragment was effective, protecting five of the eight animals in the group (62.5%) from death.  LigBrep comprises amino acid residues 1 through 628 of LigB, whose total length is 1891 residues.  Although the survival rate is nothing to get excited over, what distinguishes the LigBrep DNA vaccine from the conventional subunit vaccines tested in earlier studies is that the kidneys from 4 of the 5 survivors were culture negative, indicating that sterilizing immunity was achieved in 80% of the animals that survived infection.

Another notable outcome of the study was that protection was achieved even though the challenge strain and the vaccine's lig gene originated from different Leptospira serovars.  One of the problems with killed whole-cell vaccines is that they only protect against Leptospira serovars present in the vaccine formulation because they target LPS, whose structure varies among different serovars.  Leptospira proteins tend to be similar in amino acid sequence across different species and are therefore more attractive as vaccines.  (The "killed-whole leptospires" control plotted in the graph above was generated from the challenge strain).

So how does DNA vaccination induce sterilizing immunity against Leptospira?  As always, more studies are needed to explore this issue, but I will go ahead and speculate. DNA vaccines that are administered by standard injection stimulate a Th1-biased immune response.  Studies with cattle have suggested that vaccines must stimulate Th1 immunity to minimize kidney colonization by Leptospira (see this study, for example).  Moreover, an earlier study by Dellagostin's group demonstrated sterilizing immunity against L. interrogans in some animals immunized with a Mycobacterium bovis BCG strain that was engineered to express LipL32, the major outer membrane protein of L. interrogans.  BCG also stimulates Th1 immunity.

Why would a Th1 response be necessary for sterilizing immunity against Leptospira?  Th1 cytokines help steer B cells into producing an IgG isotype that is strongly recognized by Fc receptor on phagocytes.  Consequently, bacteria bound by these IgG molecules are engulfed by opsonophagocytosis.  During Leptospira infections, opsonophagocytosis clears spirochetes from the circulation during the antibody response, raising the possibility that opsonophagocytosis also leads to sterilizing immunity by vaccines that induce production of the "right" IgG.

Th1 cells are also necessary for cellular immunity, which enhances the killing functions of macrophages so that they can rid themselves of intracellular pathogens.  Leptospira is considered to be an extracellular pathogen.  Nevertheless, there may be a transient intracellular phase that is critical during infection.  Although intracellular Leptospira has not been observed in vivo, L. interrogans is known to survive and replicate in cultured macrophages.

References

Forster KM, Hartwig DD, Seixas FK, Bacelo KL, Amaral M, Hartleben CP, & Dellagostin OA (2013). A conserved region of leptospiral immunoglobulin-like A and B proteins as a DNA vaccine elicits a prophylactic immune response against leptospirosis. Clinical and Vaccine Immunology : CVI, 20 (5), 725-731 PMID: 23486420

Zuerner RL, Alt DP, Palmer MV, Thacker TC, & Olsen SC (2011). A Leptospira borgpetersenii serovar Hardjo vaccine induces a Th1 response, activates NK cells, and reduces renal colonization. Clinical and Vaccine Immunology : CVI, 18 (4), 684-91 PMID: 21288995

Seixas FK, da Silva EF, Hartwig DD, Cerqueira GM, Amaral M, Fagundes MQ, Dossa RG, & Dellagostin OA (2007). Recombinant Mycobacterium bovis BCG expressing the LipL32 antigen of Leptospira interrogans protects hamsters from challenge. Vaccine, 26 (1), 88-95 PMID: 18063449

Toma C, Okura N, Takayama C, & Suzuki T (2011). Characteristic features of intracellular pathogenic Leptospira in infected murine macrophages. Cellular microbiology, 13 (11), 1783-1192 PMID: 21819516


Related posts

• Is sterilizing immunity against Leptospira possible with protein subunit vaccines?
• A new attentuated leptospirosis vaccine protects hamsters from lethal infection by more than one serovar of Leptospira

Is sterilizing immunity against Leptospira possible with protein subunit vaccines?

With complete bacterial genome sequences now available, "reverse vaccinology" can be conducted to identify proteins that can function as subunit vaccines.  The "gene first" approach of reverse vaccinology relies upon computer analysis of the genome sequence to identify encoded proteins with  features common to known surface-exposed and secreted bacterial proteins.  The selected genes can then be cloned and expressed as recombinant proteins.  The proteins, which may number in the hundreds, are then purified for vaccine testing in the animal model appropriate for the bacterial pathogen.  Reverse vaccinology has been employed successfully to find protective protein antigens against Neisseria meningitidis serogroup B, Streptococcus pneumoniae, group B Streptococcus, Bacillus anthracis, Porphyromonas gingivalis, and other bacterial pathogens (reviewed in this paper).

This approach sounds straightforward but in practice may not always lead to identification of effective subunit vaccines.  An important study from Ben Adler's group down in Monash University illustrates the challenges of finding a subunit vaccine that prevents chronic Leptospira infections.  They focused on serovar Hardjo, which causes chronic infections in cattle.  They selected 263 Hardjo genes that were predicted to encode surface-exposed, secreted, or lipid-modified proteins.  Among these they successfully cloned and expressed 223 genes as 238 protein antigens in E. coli.  Some genes were expressed as two or more fragments because of their large size.  210 of the 238 (88%) aggregated into inclusion bodies during expression and had to be kept dissolved in urea during their purification.  (Strangely, the urea was not removed by dialysis prior to immunization.)  The 238 purified proteins were mixed with an aluminum hydroxide adjuvant and injected into hamsters.  169 of the 238 proteins (71%) generated an antibody response, yet none succeeded in preventing colonization of the kidneys following challenge with a Hardjo strain.

It's been hard enough to find leptospiral proteins that protect hamsters from lethal disease when tested as vaccines (see this article for a review), yet Murray and colleagues sought proteins that protected against Leptospira colonization, a more difficult endeavor that has never been achieved with subunit vaccines.  The Hardjo strain they used easily colonizes the kidneys yet fails to produce any signs of disease in hamsters.  Although several studies have demonstrated that certain versions of the LigA and LigB proteins, when administered as vaccines, protect hamsters and mice from being killed by lethal strains of Leptospira, survivors are left with infected kidneys.  A vaccine that protects against disease or death but not infection may be adequate for humans, who eventually clear the spirochetes from their kidneys even following a natural infection (assuming the disease doesn't kill them).  However, vaccinated cattle infected with Hardjo may not be able to clear the spirochetes and will continue to shed infectious Leptospira into the environment, placing the entire herd and the workers handling them at risk of infection.  Hardjo infections generally don't cause signs of disease in cattle, but they can cause fetal death and drop in milk production in cows.

The choice of adjuvant and destruction of protective conformational epitopes by urea are possible reasons for failure to find a protective antigen.  On the other hand, perhaps a different method for delivery of protein antigens into animals should been considered.  Stay tuned.

Featured paper

Murray GL, Lo M, Bulach DM, Srikram A, Seemann T, Quinsey NS, Sermswan RW, Allen A, & Adler B (2013). Evaluation of 238 antigens of Leptospira borgpetersenii serovar Hardjo for protection against kidney colonisation. Vaccine, 31 (3), 495-499 PMID: 23176980

Helpful reviews

Dellagostin, O.A., Grassmann, A.A., Hartwig, D.D., Felix, S.R., da Silva, E.F., & McBride, A.J.A. (November 2011).  Recombinant vaccines against leptospirosis.  Human Vaccines 7(11):1215-1224. DOI: 10.4161/hv.7.11.17944

Serruto, D., Serino, L., Masignani, V., & Pizza, M. (May 26, 2009).  Genome-based approaches to develop vaccines against bacterial pathogens.  Vaccine 27(25-26):3245-3250.  DOI: 10.1016/j.vaccine.2009.01.072

Is the major outer membrane lipoprotein LipL32 really exposed on the surface of Leptospira?

Here's a study that may come as a surprise to those in the leptospirosis field.  The outer membrane lipoprotein LipL32 is believed to be the dominant protein on the cell surface of pathogenic species of Leptospira.  However, according to a new PLoS One article written by Pinne and Haake at UCLA, LipL32 may not be present on the surface at all.  This is an important issue to get right because the function proposed for LipL32, attachment to the extracellular matrix during infection, assumes that the lipoprotein is exposed on the surface of the spirochete.  More importantly, a number of research groups have already committed a lot of time and resources towards generating LipL32-based vaccines, which in current formulations confer (at best) weak protection against leptospirosis in rodent models (see this review for a critical analysis of the vaccine studies).

Pinne and Haake assessed surface exposure of LipL32 by two methods.  The first involved adding proteinase K to suspensions of Leptospira to digest proteins exposed on the surface of the spirochete.  When they did this, they saw that the known surface-exposed proteins OmpL37 and OmpL47 were degraded.  On the other hand, LipL32 didn't break down at all unless the spirochetes were first lysed by boiling them in a detergent (see Western blot below).

Figure 1B from Pinne and Haake (2013).  Increasing concentrations of proteinase K (up to 150 ug/ml) were added to suspensions (first five lanes) or lysates (last five lanes) of L. interrogans.  Following incubation, LipL32 was examined in a Western blot.  Source.

They next added LipL32 antibodies to Leptospira to see if they bound to the surface of the spirochete.  They did not, providing additional evidence that LipL32 was not exposed on the cell surface.  The authors tested LipL32 antibodies from different sources in an attempt to rule out the  possibility that failure of antibody binding was due to the surface-exposed portion of LipL32 not being antigenic.  LipL32 antiserum raised in rabbits, monoclonal LipL32 antibodies raised in mice, and LipL32 antibodies purified from the sera of leptospirosis patients all failed to bind the surface of Leptospira unless the outer membrane was chemically (with methanol or EDTA) or physically disrupted.  Note that antibodies raised against OmpL54, a known suface-exposed protein, reacted strongly with intact Leptospira (last pair of images below).

Figure 3A from Pinne and Haake (2013).  Bound antibody was detected with a secondary fluorescent antibody (green).  The spirochetes were also stained with DAPI, a penetrating dye that stains DNA (blue).  Left column, intact Leptospira, right column, methanol-treated Leptospira. Source.

In light of these results, the authors took another look at the 2005 study by Cullen and coauthors, who claimed LipL32 was surface exposed.  In contrast to Pinne and Haake, Cullen and colleagues detected binding of LipL32-specific antibodies to intact Leptospira in three different assays.  However, Pinne and Haake point out that antibody binding in their assays was extremely weak when  the abundance of LipL32 is considered.  The most striking example was the immunoelectron microscopy image of Leptospira treated with gold-labeled LipL32 antibody (see next image).  Yes, the surface ended up labeled, with a mean of 10.8 gold particles per spirochete cell.  However, we now know that there are 38,000 copies of LipL32 in each bacterial cell, making LipL32 the most abundant protein of L. interrogans (see this blog post about the Leptospira protein census).  If LipL32 were really surface exposed, the surface of the spirochete should have been packed with gold particles.

Figure 5 from Cullen et al. (2005).

Cullen and colleagues also mixed suspensions of Leptospira with a biotin probe that reacts with primary amines (mostly on lysine side chains).  The probe should have reacted solely with surface-exposed proteins since it's unable to penetrate the outer lipid bilayer and is assumed to be too large to diffuse through outer membrane porins.  Biotinylated proteins were separated by two-dimensional electrophoresis and identified by mass spectrometry (see next figure).  Although LipL32 was one of the few proteins labeled with biotin, it's hard to make a firm conclusion about its surface exposure because proteins known to be located underneath the outer membrane, FlaB1 (a flagellar protein) and GroEL (a cytoplasmic heat shock protein), were also labeled with biotin. It's possible that LipL32 was labeled despite being located underneath the surface because the membrane was damaged while the spirochetes were being harvested for the experiment.

Modified from Figure 2 of Cullen et al., 2005.  Biotinylated proteins were separated by two-dimnesional electrophoresis.  Spots were removed and analyzed by mass spectrometry to identify proteins.  Multiple spots for each protein in the result of members of the population of each protein reacting with different numbers of biotin molecules.  "LipL32.16" was generated by proteolysis of LipL32.

Based on the intense labeling of LipL32 with biotin, Cullen and coauthors declared LipL32 the most abundant protein on the cell surface.  They speculated that LipL32 is poorly accessible to large molecules such as antibodies and proteases (which in their study failed to digest any protein when added to intact Leptospira) because the LPS side chains act as a "rainforest canopy" that can be penetrated only by smaller molecules such as biotin. This is a reasonable supposition because LipL32 is up to 60Å in length, whereas the distance between the outer membrane and the surface of the LPS layer is 92Å, according to a cryoelectron microscopy study of L. interrogans.  On the other hand, Pinne and Haake concluded that LipL32 is entirely or almost entirely subsurface since their assays failed to detect even a hint of the lipoprotein on the surface of Leptospira.  They maintain that the reactivity of surface probes with LipL32 observed by Cullen and colleagues was an artifact generated by the presence of damaged spirochetes in their assays and the massive copy number of LipL32. 

The results from Pinne and Haake's study do not rule out the "rainforest canopy" model since they did not test smaller surface probes that could penetrate into the LPS side chain layer.  Additional studies are needed to pin down the location of LipL32 relative to the surface of Leptospira.

References

Pinne, M., & Haake, D.A. (2013). LipL32 is a subsurface lipoprotein of Leptospira interrogans: Presentation of new data and reevaluation of previous studies. PLoS ONE, 8 (1) DOI: 10.1371/journal.pone.0051025

Cullen, P.A., Xu, X., Matsunaga, J., Sanchez, Y., Ko, A.I., Haake, D.A., & Adler, B. (2005). Surfaceome of Leptospira spp. Infection and Immunity, 73 (8), 4853-4863 DOI: 10.1128/IAI.73.8.4853-4863.2005

Related posts

• Protein census of Leptospira interrogans
• The major outer membrane protein of Leptospira interrogans: Not essential for infection?

LigB of Leptospira interrogans: Avoiding or exploiting complement?

LigB has turned out to be a versatile surface protein for Leptospira interrogans.  The protein is one of several that the spirochete uses to stick to the extracellular matrix, a critical step in colonizing host tissues.  In addition, LigB's ability to bind fibrinogen may help L. interrogans spread within the host by slowing clot formation.  According to separate studies from the U.S. and Brazil published last year, LigB also helps L. interrogans fend off attack by the host complement system.

What's the evidence that LigB protects Leptospira from complement?  L. interrogans, like many pathogens, survives even when complement is present.  Its resistance to complement is reflected by its ability to survive in human serum, which contains all the complement components necessary to assemble the deadly membrane attack complex within exposed microbial membranes.  On the other hand, the nonpathogen Leptospira biflexa, which lacks the ligB gene, is rapidly killed by human serum.  The U.S. study demonstrated that transformation of L. biflexa with a plasmid expressing LigB allowed the nonpathogen to survive in human serum diluted to 5%, a concentration that easily killed L. biflexa harboring a similar plasmid lacking ligB.

How exactly does LigB protect Leptospira from the onslaught of complement?  A defensive strategy deployed by many pathogens is to seize host complement regulators, which are present to prevent complement activation on host cells.  The Brazilian study showed that LigB grabs several complement regulators that diminish the levels of the key complement proteins C3b and C4b on the bacterial surface.  Two of these regulators are the factor H protein and the C4-binding protein (C4BP).  Both complement regulators break apart the C3 convertases, the enzyme complexes that generate C3b from C3.  Factor H and C4BP also serve as cofactors for the protease factor I, which cleaves C3b and C4b into smaller pieces to prevent their assembly into the C5 convertase.  The C5 convertase is what triggers assembly of the membrane attack complex.  As expected, the investigators found that the breakdown of C3b and C4b by factor I in the presence of its complement regulators was accelerated when LigB was present.

LigB is not the only Leptospira protein that captures complement regulators.  The proteins LenA (originally called LfhA) and LcpA bind factor H and C4BP, respectively.  The importance of factor H in protecting L. interrogans can be seen in the bar graph below.  Survival of L. interrogans was poor in serum lacking factor H.  Addition of factor H to the serum up to the concentration found in blood (500 μg/ml) enhanced survival of the spirochete.

Figure 1 from Castiblanco-Valencia et al. (2012).  Survival of L. interrogans in factor H-depleted serum with 500 μg/ml factor H (FH) is set to 100%.

Surprisingly, the U.S. study revealed that a small segment of LigB grabbed C3b and C4b.  Why would L. interrogans risk capturing complement proteins while simultaneously collecting complement regulators that inactivate those same proteins?  Other pathogens do just fine capturing complement regulators without actively grabbing complement components.  It's possible that C3b and C4b are inactivated more effectively by the complement regulators when all components are bound to LigB.

There's another possibility that should be considered.  What went unmentioned in both papers is that C3b and its cleavage products, which may remain attached to the bacterial surface, are opsonins recognized by phagocytes aiming to grab and engulf microbial intruders.  However, several intracellular pathogens use complement receptors as an entry point to invade macrophages.  L. interrogans has been shown to survive within cultured macrophages and even remained intact within macrophages in a zebrafish model, as I've explained in another post.  Is it possible that L. interrogans uses C3b to grab and invade macrophages?

Featured papers

Castiblanco-Valencia MM, Fraga TR, da Silva LB, Monaris D, Abreu PAE, Strobel S, Jozsi M, Isaac L, & Barbosa AS (2012). Leptospiral immunoglobulin-like proteins interact with human complement regulators factor H, FHL-1, FHR-1, and C4BP. Journal of Infectious Diseases, 205 (6), 995-1004 DOI: 10.1093/infdis/jir875

Choy H (2012). Multiple activities of LigB potentiate virulence of Leptospira interrogans: Inhibition of alternative and classical pathways of complement. PLoS ONE, 7 (7) DOI: 10.1371/journal.pone.0041566

Other helpful papers

Verma A, Hellwage J, Artiushin S, Zipfel PF, Kraiczy P, Timoney JF, and Stevenson B (March 2006).  LfhA, a novel factor H-binding protein of Leptospira interrogans.  Infection and Immunity 74(5):2659-2666.  Link

Barbosa AS, Monaris D, Silva LB, Morais ZM,  Vasconcellos SA,  Cianciarullo AM, Isaac L, and Abreu PAE (July 2010).  Functional characterization of LcpA, a surface-exposed protein of Leptospira spp. that binds the human complement regulator C4BP.  Infection and Immunity 78(7):3207-3216.  DOI: 10.1128/IAI.00279-1010.1128/IAI.00279-10

Related posts

• A tick protein helps the Lyme disease spirochete fight complement
• Zebrafish model of leptospirosis: Where's the relevance?

Spirochete research: 2012 in review

Here are some of my favorite spirochete papers from 2012.  Direct access to all research articles (some behind a paywall) is provided via the DOI links.  Where present, the links above the citations lead to my blog posts about the studies.

PATHOGENESIS

Two distinct regions of the Borrelia burgdorferi BBK32 lipoprotein sequentially mediate binding to the vessel wall in vivo during escape of the spirochete from the bloodstream.

• Moriarty TJ, Shi , Lin Y-P, Ebady R, Zhou H, Odisho T, Hardy P-O, Salman-Dilgimen A, Wu J, Weening EH, Skare JT, Kubes P, Leong J, and Chaconas G (December 2012).  Vascular binding of a pathogen under shear force through mechanistically distinct sequential interactions with host macromolecules.  Molecular Microbiology 86(5):1116-1131.  DOI: 10.1111/mmi.12045

The Leptospira interrogans LigB protein protects the spirochete from complement by capturing complement regulatory proteins.

• Castiblanco-Valencia MM, Fraga TR, da Silva LB, Monaris D, Abreu PAE, Strobel S, Jozsi M, Isaac L, and Barbosa AS (March 15, 2012).  Leptospiral immunoglobulin-like proteins interact with human complement regulators factor H, FHL-1, FHR-1, and C4BP.  The Journal of Infectious Diseases 205(6):995-1004.  DOI: 10.1093/infdis/jir875
• Choy HA (July 2012).  Multiple activities of LigB potentiate virulence of Leptospira interrogans: inhibition of alternative and classical pathways of complement.  PLoS One 7(7):e41566. DOI: 10.1371/journal.pone.0041566

A B. burgdorferi lipase with hemolytic activity in vitro:

• Shaw DK, Hyde JA, and Skare JT (January 2012).  The BB0646 protein demonstrates lipase and haemolytic activity associated with Borrelia burgdorferi, the aetiological agent of Lyme disease.  Molecular Microbiology 83(2):319-334.  DOI: 10.1111/j.1365-2958.2011.07932.x

TRANSMISSION

Borrelia burgdorferi needs the alternative sigma factor RpoS to flee from the tick's midgut

• Dunham-Ems SM, Caimano MJ, Eggers CH, and Radolf JD (February 2012).  Borrelia burgdorferi requires the alternative sigma factor RpoS for Dissemination within the vector during tick-to-mammal transmission.  PLoS Pathogens 8(2):e1002532.  DOI: 10.1371/journal.ppat.1002532

MOTILITY

Video microscopy of B. burgdorferi swimming around in gelatin and mouse tissue:

• Harman MW, Dunham-Ems SM, Caimano MJ, Belperron AA, Bockenstedt LK, Fu HC, Radolf JD, and Wolgemuth CW (February 21, 2012).  The heterogeneous motility of the Lyme disease spirochete in gelatin mimics dissemination through tissue.  Proceedings of the National Academy of Sciences USA 109(8):3059-3064.  DOI: 10.1073/pnas.1114362109

L. interrogans sheath protein homologs that are not needed for flagellar sheath formation:

• Lambert A, Picardeau M, Haake DA, Sermswan RW, Srikram A, Adler B, and Murray GA (June 2012).  FlaA proteins in Leptospira interrogans are essential for motility and virulence but are not required for formation of the flagellum sheath.  Infection and Immunity 80(6):2019-2025.  DOI: 10.1128/IAI.00131-12

ULTRASTRUCTURE

A close look at the ultrastructure of Leptospira without the artifacts generated by conventional electron microscopy:

• Raddi G, Morado DR, Yan J, Haake DA, Yang XF, and Liu J (March 2012).  Three-dimensional structures of pathogenic and saprophytic Leptospira species revealed by cryo-electron tomography.  Journal of Bacteriology 194(6):1299-1306.  DOI: 10.1128/JB.06474-11

METAL TOXICITY

B. burgdorferi BicA, a protein that protects the spirochete from the toxic effects of copper and iron:

• Wang P, Lutton A, Olesik J, Vali H, and Li X (December 2012).  A novel iron- and copper-binding protein in the Lyme disease spirochaete.  Molecular Microbiology 86(6):1441-1451.  DOI: 10.1111/mmi.12068

ANTIBIOTIC THERAPY

Inflammatory spirochete debris left behind following antibiotic treatment for Lyme disease

• Bockenstedt LK, Gonzalez DG, Haberman AM, and Belperron AA (July 2, 2012).  Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy.  The Journal of Clinical Investigation 122(7):2652-2660.  DOI: 10.1172/JCI58813

A critical analysis of a study that demonstrated persistence of B. burgdorferi in infected rhesus monkeys that were treated with antibiotics:

• Wormser GP, Baker PJ, O'Connell S, Pachner AR, Schwartz I, and Shapiro ED (July 2012).  Critical analysis of treatment trials of rhesus macaques infected with Borrelia burgdorferi reveals important flaws in experimental design.  Vector-borne and Zoonotic Diseases 12(7):535-538.  DOI: 10.1089/vbz.2012.1012
• Embers ME, Barthold SW, Borda JT, Bowers L, Doyle L, Hodzic E, Jacobs MB, Hasenkampf NR, Martin DS, Narasimhan S, Phillippi-Falkenstein KM, Purcell JE, Ratterree MS, and Philipp MT (January 2012).  Persistence of Borrelia burgdorferi in rhesus macaques following antibiotic treatment of disseminated infection.  PLoS One 7(1):e29914.  DOI: 10.1371/journal.pone.0029914

A tale of two more studies: topical antibiotics applied to tick bites to prevent Lyme disease

• Wormser GP, Daniels TJ, Bittker S, Cooper D, Wang G, and Pavia CS (March 15, 2012).  Failure of topical antibiotics to prevent disseminated Borrelia burgdorferi infection following a tick bite in C3H/HeJ mice.  The Journal of Infectious Diseases 205(6):991-994.  DOI: 10.1093/infdis/jir382

DIAGNOSTICS

Not so golden?  Microscopic agglutination test for diagnosis of leptospirosis

• Limmathurotsakul D, Turner EL, Wuthiekanun V, Thaipadungpanit J, Suputtamongkol Y, Chierakul W, Smythe LD, Day NPJ, Cooper B, and Peacock SJ (August 1, 2012).  Fool’s gold: Why imperfect reference tests are undermining the evaluation of novel diagnostics: A reevaluation of 5 diagnostic tests for leptospirosis.  Clinical Infectious Diseases 55(3):322-331.  DOI: 10.1093/cid/cis403

ECOLOGY

Do nonspiral spirochetes help clean our environment?

• Caro-Quintero A, Ritalahti KM, Cusick KD, Loffler FE, and Konstandtinidis KT (May/June 2012).  The chimeric genome of Sphaerochaeta: Nonspiral spirochetes that break with the prevalent dogma in spirochete biology.  mBio 3(3):e00025-12.  DOI: 10.1128/mBio.00025-12
• Ritalahti KM, Justicia-Leon SD, Cusick KD, Ramos-Hernandez N, Rubin M, Dornbush J, and Loffler FE (January 2012).  Sphaerochaeta globosa gen. nov., sp. nov. and Sphaerochaeta pleomorpha sp. nov., free-living, spherical spirochetes.  International Journal of Systematic and Evolutionary Microbiology 62(Pt 1):210-216.  DOI: 10.1099/ijs.0.023986-0

 

BIOFILM

Biofilms of the Lyme disease spirochete

• Sapi E, Bastian SL, Mpoy CM, Scott S, Rattelle A, Pabbati N, Poruri A, Burugu D, Theophilus PAS, Pham TV, Data A, Dhaliwal NK, MacDonald A, Rossi MJ, Sinha SK, and Luecke DF (October 2012).  Characterization of biofilm formation by Borrelia burgdorferi in vitro.  PLoS One 7(10):e48277.  DOI: 10.1371/journal.pone.0048277

 

HISTORY

Looking for the syphilis spirochete in ancient bones

• Montiel R, Solorzano E, Diaz N,  Alvarez-Sandoval BA, Gonzalez-Ruiz M, Canadas MP, Simoes N, Isidro A, and Malgosa A (May 2012).  Neonate human remains: A window of opportunity to the molecular analysis of syphilis.  PLoS One 7(5):e36371.  DOI: 10.1371/journal.pone.0036371

Presenting flawed studies directly to the public to bypass the scientific peer-review process:

• Armelagos GJ, Zuckerman MK, and Harper KN (March 2012).  The science behind pre-Columbian evidence of syphilis in Europe: research by documentary.  Evolutionary Anthropology 21(2):50-57.  DOI: 10.1002/evan.20340

REVIEWS

Here are two excellent review articles that appeared during the past year:

• Radolf JD, Caimano MJ, Stevenson B, and Hu LT (February 2012).  Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes.  Nature Reviews Microbiology 10(2):87-99.  DOI: 10.1038/nrmicro2714
• Charon NW, Cockburn A, Li C, Liu J, Miller KA, Miller MR, Motaleb MA, and Wolgemuth CW (2012).  The unique paradigm of spirochete motility and chemotaxis.  Annual Reviews of Microbiology 66:349-370.  DOI: 10.1146/annurev-micro-092611-150145

Not so golden? Microscopic agglutination test for diagnosis of leptospirosis

The microscopic agglutination test (MAT) is designated the "gold standard" for the laboratory diagnosis of leptospirosis, a spirochete disease that can cause severe illness if not promptly treated.  Although imperfect, MAT is used as the benchmark when the performance of another diagnostic test for leptospirosis is being assessed.  It is also used to determine the prevalence of leptospirosis in a population.  How imperfect is MAT?  A recent study by Limmathurotsakul and colleagues, published in the journal Clinical Infectious Diseases, claims that its performance is much worse than scientists previously thought.

MAT involves mixing serial dilutions of patient sera with live suspensions of Leptospira.  If agglutinating antibodies against Leptospira are present, the spirochetes will clump.  The clumps can be seen by darkfield microscopy.  Although the idea behind MAT is simple to understand, the technique itself is cumbersome.  Since agglutinating antibodies react best with the specific Leptospira serovar infecting the patient, cultures of at least one serovar from each of the ~20 major Leptospira serogroups must be maintained.  To perform the assay, each serum dilution is mixed individually with a suspension from each culture and examined by microscopy one at a time.  The assay is time consuming, laborious, and potentially hazardous to laboratory personnel.  For these reasons MAT is not routinely employed for diagnostic testing outside of the research setting.

The performance of a diagnostic test is judged by its sensitivity and specificity.  The problem with leptospirosis is figuring out how many actually have the disease so that the sensitivity can be calculated accurately.  Since the sensitivity of culture is poor, researchers rely on antibody tests such as MAT to identify leptospirosis cases.  This approach assumes that the sensitivity and specificity of MAT are 100%.

In general there are two problems with using antibody tests for diagnostics.  The first is that it takes time for the immune system to generate enough antibody that can be detected.  The second problem is that those with previous exposure to the pathogen will test positive even if they are not currently infected.  To minimize these problems, patients with the signs and symptoms of leptospirosis are deemed to have a positive MAT if they fulfill one of the following criteria.

• At least a four-fold increase in MAT titer between paired sera. 
• At least a 1:400 MAT titer when only a single specimen is available.  This cutoff is sometimes adjusted based on the prevalence of leptospirosis in the population being examined.

Since some false negative MAT cases can be identified by culture, one way to calculate the sensitivity of MAT is to add the number of MAT-positive and culture-positive (but MAT-negative) cases together to estimate the number of patients with leptospirosis and then calculate the percentage of MAT-positive cases among these patients.  Limmathurotsakul and colleagues performed these calculations with data from their four earlier studies conducted in Thailand.  A total of 413 patients tested positive by MAT or culture (or both).  They found that the sensitivity of MAT was 86%-96% across the four sets of data.  The remaining 4-14% were false negatives, having tested positive by culture but not by MAT.

The authors next calculated the true sensitivity and specificity of MAT with a statistical tool called latent class analysis, which does not assume any perfect gold standard.  Since there is no perfect test, the true disease status of each patient is the unknown or "latent" variable.  Results from multiple diagnostic tests are related to the latent variable using statistical models.  The calculations go beyond the scope of this blog post, but the bottom line is that the true sensitivity and specificity of each diagnostic test can be estimated with these models.  In addition to MAT and culture, the authors tested some of their patients with an immunofluorescence assay (IFA), lateral flow test (LF), and/or PCR. Latent class analysis is more powerful when the diagnostic tests being evaluated detect different features of the infection.  MAT, IFA, and LF are antibody tests, and culture and PCR detect the pathogen itself.

The sensitivity of MAT calculated by this method turned out to be only 49.8%, much lower than the 86%-96% calculated using the standard method that assumes a perfect gold standard.  The sensitivity of culture alone was 10.5%.  Combining culture with MAT did not help much; the sensitivity of the combined approach was only 55.5%.  The low sensitivity of "MAT plus culture" suggests that the specificities calculated for the alternative tests may be underestimated by the standard method.  This is because some of the many false-negative cases may be correctly identified as having leptospirosis by the alternative tests.  This turned out to be the case for two of the tests.   Specificities for all tests were over 95% by latent class analysis.  However, the specificities for PCR (82.5%) and the lateral flow test (70.5%) were lower when "MAT plus culture" was assumed to be the perfect gold standard.

You can see that the accuracy of alternative leptospirosis tests is underestimated when MAT (or MAT plus culture) is assumed to be the perfect reference test.  Another implication of the study is that the prevalence of leptospirosis has been underestimated, at least in Thailand.  The only other study to evaluate the performance of MAT by latent class analysis was conducted by the CDC here in the U.S almost a decade ago.  In contrast to the Limmathurostsakul study, the CDC study determined that the sensitivity of MAT was high, at 98.2%.  There were many differences between the two studies, including the patient population, the alternative tests evaluated, the time interval between collection of paired sera, and the number of serovars included for MAT.  The poor performance of MAT in the Thailand study may therefore not be a universal finding.

References

Limmathurotsakul D, Turner EL, Wuthiekanun V, Thaipadungpanit J, Suputtamongkol Y, Chierakul W, Smythe LD, Day NPJ, Cooper B, Peacock SJ (August 2012).  Fool's gold: why imperfect reference tests are undermining the evaluation of novel diagnostics: a reevaluation of 5 diagnostic tests for leptospirosis.  Clinical Infectious Diseases 55(3):322-331.  DOI: 10.1093/cid/cis403

Bajani MD, Ashford DA, Bragg SL, Woods CW, Aye T, Spiegel RA, Plikaytis BD, Perkins BA, Phelan M, Levett PN, and Weyant RS (February 2003).  Evaluation of four commercially available rapid serologic tests for diagnosis of leptospirosis.  Journal of Clinical Microbiology 41(2):803-809.  DOI:  10.1128/JCM.41.2.803-809.2003

A new attenuated leptospirosis vaccine protects hamsters from lethal infection by more than one serovar of Leptospira

Scientists have demonstrated that a new attenuated leptospirosis vaccine protects laboratory hamsters from being killed by Leptospira, even when the challenge and vaccine strains belong to different serovars (immune types).^1,2  This is the first leptospirosis vaccine to confer complete cross-protection against lethal infection by a serovar different from the one used for immunization.

The leptospirosis vaccines that are out on the market are still formulated with killed Leptospira or sometimes their outer membrane.  These traditional vaccines are administered primarily to dogs, cattle, and pigs.  Human leptospirosis vaccines are not available in most countries, even in areas where leptospirosis is endemic.

New types of leptospirosis vaccines are needed since the traditional killed vaccines are flawed.  One problem is that immunity is serovar specific.  For this reason a vaccine must contain all the serovars that the target population may encounter.  Even when the vaccine manufacturers figure out which serovars are circulating, a new serovar may emerge, rendering the vaccine ineffective as the new serovar spreads through the susceptible population.  The vaccine must then be reformulated at substantial cost.

This is exactly what happened to the leptospirosis vaccines that are given to dogs.³  The early canine vaccines, first available in the 1970s, contained the serovars Canicola and Icterohemorrhagiae.  These vaccines worked fine until the late 1980s or so, when new serovars started to appear in infected dogs, even in those that had been vaccinated.  Since then vaccine makers have added the serovars Grippotyphosa and Pomona to their vaccines.  Nevertheless with over 200 pathogenic serovars of Leptospira lurking out there, we don't know when or which additional serovars will emerge in the future.

It would be nice to have a single leptospirosis vaccine formulation that would protect against all serovars.  The protective effect of traditional vaccines is due to antibodies generated against lipopolysaccharide (LPS), whose structure differs among the serovars of Leptospira.  Since immunization elicits antibodies that recognize the LPS of only the serovars included in the vaccine, vaccinated individuals remain susceptible to infection by other serovars.

To get around this problem, scientists have been testing individual Leptospira surface proteins as potential vaccines in rodent models of leptospirosis.  Leptospira surface proteins tend to be antigenically conserved among the different serovars:  antibodies generated against a protein from one serovar often reacts against the same protein expressed by other serovars.  According to many studies the LipL32 and Lig surface lipoproteins, when delivered as recombinant proteins, naked DNA, or by microbial vectors (adenovirus and Mycobacterium bovis), apparently protected hamsters or guinea pigs from lethal infection by Leptospira.  Unfortunately one of the leaders in the leptospirosis field, Ben Adler (also an author of the two featured papers), has questioned the interpretation of these studies.⁴  He points out that the challenge strains used in several studies were not sufficiently lethal, making it easier to observe a protective effect of the vaccine.  Moreover some studies claimed statistically significant protective effects of the protein vaccine when in fact there was none upon Adler's reanalysis of the data.  The only protein to convincingly exert a protective effect in an appropriate animal model was LigA⁵ although the ability of the LigA subunit vaccine to cross-protect against different serovars of Leptospira has yet to be tested.  However there is one major problem with using LigA as a vaccine--not all Leptospira strains have the ligA gene.⁶

In the two studies described here the investigators took a step back from looking at individual proteins and developed an attenuated strain to use for immunization.  The properties of the attenuated strain, designated M1352, are described in the paper authored by Murray and colleagues.²  The M1352 strain was not developed by the classic approach of continuously growing and passaging the bacteria in culture until they lost their ability to cause disease.  Instead the strain was one of a large collection of mutants generated by random transposon mutagenesis of L. interrogans serovar Manilae.  The M1352 strain had the transposon inserted in a gene located in a large cluster of genes encoding enzymes that assemble LPS.  The mutation had subtle effects on the reactivity of M1352 with various antibodies raised against leptospiral LPS, suggesting that the LPS structure itself was somehow changed in M1352 when compared with the wild-type Manilae strain.

Since LPS is a crucial surface component that interacts with the host, it was not too surprising that M1352 was not able to cause lethal infections like its wild-type Manilae parent.  When they infected hamsters with the M1352 strain, the spirochetes were unable to kill the hamsters or even establish an infection in the kidneys.  Despite the efficient clearance of M1352, the Leptospira lingered long enough in the hamsters to provoke an antibody response. Western blots of L. interrogans lysates revealed strong reactivity of antibodies from the M1352-infected hamsters to a number of proteins. Because the M1352 strain generated an antibody response without establishing an infection, the authors decided to test the weakened strain as a vaccine in the hamster model in a follow-up study.¹

In the second study, Srikram and colleagues¹ demonstrated that immunization of hamsters with a single dose of live M1352 was more effective than a dose of heat-killed wild-type strain in protecting hamsters from being killed by the wild-type Manilae strain. The M1352 vaccine also did a better job in preventing colonization of the kidneys by the spirochete and in minimizing lung hemorrhage than the heat-killed vaccine.

When they challenged the vaccinated hamsters with a different serovar, a Pomona strain, all the hamsters immunized with live M1352 survived whereas 60% of animals immunized with heat-killed wild-type Manilae perished.  However the M1352 vaccine didn't work perfectly.  Although all hamsters immunized with live M1352 survived the challenge with the Pomona strain, the kidneys from 90% of the animals were culture positive, and 90% had hemorrhaged lungs.  Nevertheless this is the first time that complete protection from death was observed following challenge of vaccinated animals by a serovar unrelated to the vaccine strain.  They also showed that the M1352 vaccine had to be administered alive.  Heat-killed or chemically-killed M1352 vaccine failed to protect hamsters from lethal infection.

The investigators next tried to figure out which component of the M1352 strain was the protective cross-reactive antigen targeted by the hamster's immune system.  They wondered whether the live M1352 and heat-killed wild-type Manilae vaccines generated antibody responses to different proteins.  When they probed separate two-dimensional blots of L. interrogans membrane preparations of serovar Pomona with antibodies from hamsters immunized with M1352 and heat-killed wild-type Manilae, a number of protein spots lit up.  Most proteins, including LipL32, reacted with both sets of antibodies.  These proteins are unlikely to account for the cross-protection conferred by the M1352 vaccine since the presence of these antibodies in the hamsters immunized with heat-killed Manilae failed to protect the animals from being killed by the Pomona strain.  On the other hand, four Pomona proteins were recognized only by hamsters receiving the attenuated vaccine:

• Loa22, the only surface protein known to be essential for L. interrogans to cause lethal infections⁷
• a homolog of GspG, a component of the type II secretion system
• LA1939, a possible lipoprotein of unknown function
• OmpL36, a surface-exposed outer membrane protein of unknown function⁸

Since GspG is not a surface component of other bacteria and nothing is known about where LA1939 is located on Leptospira, Loa22 and OspL36 are the best candidates to test as potential cross-protective vaccines.

Featured papers

1. Srikram A, Zhang K, Bartpho T, Lo M, Hoke DE, Sermswan RW, Adler B, and Murray GL (March 15, 2011).  Cross-protective immunity against leptospriosis elicited by a live, attenuated lipopolysaccharide mutant.  Journal of Infectious Diseases 203(6):870-879.  DOI: 10.1093/infdis/jiq12


2. Murray GL, Amporn S, Henry R, Hartskeerl RA, Sermswan RW, and Adler B (November 2010).  Mutations affecting Leptospira interrogans lipopolysaccharide attenuate virulence.  Molecular Microbiology 78(3): 701-709.  DOI: 10.1111/j.1365-2958.2010.07360.x

Helpful references

3. Guerra MA (February 15, 2009).  Leptospirosis. Journal of the American Veterinary Medical Association 234(4):472-478.  DOI: 10.2460/javma.234.4.472

4. Adler B and de la Pena Moctezuma (January 27, 2010).  Leptospira and leptospirosis.  Veterinary Microbiology 140(3-4):287-296.  DOI: 10.1016/j.vetmic.2009.03.012

5. Silva, ÉF, Medeiros MA, McBride AJA, Matsunaga J, Esteves GS, Ramos JGR, Santos CS, Croda J, Homma A, Dellagostin OA, Haake DA, Reis MG, and Ko AI (August 14, 2007).  The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis. Vaccine 25(33):6277-6286.  DOI: 10.1016/j.vaccine.2007.05.053

6. McBride AJA, Cerqueira GM, Suchard MA, Moreira MA, Zuerner RL, Reis MG, Haake DA, Ko AI, and Dellagostin OA (March 2009). Infection, Genetics and Evolution 9(2):196-205.  DOI: 10.1016/j.meegid.2008.10.012

7. Ristow P, Bourhy P, da Cruz McBride FW, Figueira CP, Huerre M, Ave P, Girons IS, Ko AI, and Picardeau M (July 2007).  The OmpA-like protein Loa22 is essential for leptospiral virulence. PLoS Pathogens3(7):e97. DOI: 10.1371/journal.ppat.0030097

8.Pinne M and Haake DA (June 2009).  A comprehensive approach to identification of surface-exposed, outer membrane-spanning proteins of Leptospira interrogans.  PLoS One 4(6):e6071. DOI: 10.1371/journal.pone.0006071