Saturday, April 23, 2016

Long-term antibiotics for those with chronic symptoms that may or may not be related to Lyme disease

A Lyme disease study published a few weeks ago in the New England Journal of Medicine has received a lot of coverage in the press.  According to the abstract of the study, Berende and colleagues conducted a randomized placebo-controlled clinical trial to test the effectiveness of long-term "longer-term" antibiotics in treating "longer-term" chronic symptoms "attributed" to Lyme disease.

As many readers of this blog know, treatment of Lyme disease is a controversial topic.  Antibiotics are effective in treating Lyme disease, but 10-20% experience symptoms such as fatigue, muscular aches, and joint pain for at least 6 months following conventional treatment.  The cause of the persisting symptoms is not known.  They could be due to tissue damage caused by the infection, ongoing inflammation, or bacteria that survived antibiotic treatment.  Mainstream medical societies such as the IDSA do not believe that lingering infection is responsible for the persisting symptoms, and they do not recommend retreatment with antibiotics.  Four randomized controlled studies conducted in the U.S. showed little benefit of retreating these patients with antibiotics for up to 3 months.  On the other hand, not-so-mainstream groups such as ILADS dispute the interpretation of the data.  They insist that the treatment groups did show some improvement and that longer treatment regimens lasting longer than 3 months are needed for complete recovery of these patients.

There is another group of patients that also suffer from enduring fatigue, muscle aches, and joint pain.  They may or may not have had Lyme disease in the past, but their ongoing symptoms stem from some other condition.  Unfortunately, they may be misdiagnosed with "chronic Lyme disease" and end up being treated for a long time with antibiotics in an attempt to eradicate an infection that they don't have.

The new NEJM paper describes a randomized placebo-controlled trial that was conducted in the Netherlands.  281 subjects who had been experiencing chronic symptoms blamed on Lyme disease (fatigue, muscle aches, joint pain) were randomized into three groups.  All three groups were treated with ceftriaxone intravenously for two weeks.  The subjects were next given oral antibiotics or placebo for 12 weeks.  One group was treated with doxycycline, another group with both clarithromycin and hydroxychloroquine, and the third group was given placebo pills.

The graph below shows that the physical quality of life, the primary outcome measure, improved a little for all groups.  Because there was no difference in outcome among the three groups, the authors concluded that longer-term antibiotics were no better than short-term antibiotics in alleviating symptoms.  We don't know whether the initial two-week treatment with ceftriaxone had anything to do with the slight improvement since there was no true placebo (antibiotic-free) group.

Change in mean SF-36 physical component summary scores before and after treatment period.  Figure 2 from Berende et al., 2016.

Why wasn't a true placebo group?  The authors worried about withholding antibiotics from subjects who might have an infection that should be treated.  11% of the subjects hadn't been treated with antibiotics for their symptoms prior to their acceptance into the study.  This wasn't an issue with the earlier U.S. trials since previous treatment of Lyme disease with antibiotics was a requirement for acceptance into those studies, which included true placebo groups.

One baffling aspect of the study was the inclusion of subjects who might not have had Lyme disease prior to the appearance of their chronic symptoms. Only a third of the subjects had objective clinical features of Lyme disease (erythema migrans, meningoradiculitis, or acrodermatitis chronica atrophicans) immediately preceding their chronic symptoms, and a little more than a half recalled a tick bite.  Contrast this with the earlier U.S. studies, which only accepted patients with chronic symptoms that followed antibiotic treatment of a well-documented case of Lyme disease.

The remaining subjects did not have any objective features of Lyme disease before their chronic symptoms appeared.  The only evidence of a previous episode of Lyme disease was positive antibody testing by Western blot.  However, the antibodies may have been elicited by a Borrelia infection in the distant past.  Their past episode of Lyme disease may not be related to their chronic symptoms, which aren't specific for Lyme disease.  Another problem with relying solely on Western blots to diagnose Lyme disease is that false positives occur.  Without additional evidence, it is hard to be sure that their chronic symptoms were related to Lyme disease.

Nevertheless, the editorial accompanying the paper expressed support for the relaxed inclusion criteria used to select the subjects for the study:
Critics may rightly say that this trial does not truly capture with certainty the consequences of bona fide Lyme disease. However, studies with more stringent inclusion criteria have already been conducted, and the approach used by Berende and colleagues probably reflects the common practice in ambulatory care settings, in which patient presentations of fatigue or nonspecific pain prompt serologic checks for Lyme disease, despite evidence suggesting that these tests will not identify a probable cause or result in a treatment benefit.
The study population may reflect what's encountered by clinicians in the real world, but for a clinical trial it doesn't seem right to lump those whose chronic problems followed a real episode of Lyme disease with those whose issues had nothing to do with Lyme.  Any benefit of the antibiotics experienced by those who had genuine Lyme disease (assuming that there was any benefit) may have been obscured by the lack of benefit in those whose chronic symptoms aren't related to Lyme disease.

Berende and colleagues also defended the length of treatment, which is considered to be on the short end by those who support lengthy courses of antibiotic therapy: may be argued that 14 weeks of treatment is insufficient to show a beneficial treatment effect. However, whereas prolonged antimicrobial treatment is not uncommon for various infectious diseases, the purpose of prolonged therapy for such diseases is for the prevention of microbiologic relapse rather than for a delayed onset of clinical alleviation of signs or symptoms. We are not aware of any infectious disease in which the initial effect on signs, symptoms, and laboratory findings is delayed beyond the first 3 months of effective therapy.
But the graph above clearly shows that the subjects felt better following the treatment period.  Unfortunately, as I mentioned earlier, the improvement in the quality of life can't be attributed to the antibiotics because there was no true placebo group.

With these issues, I'm not sure how this study got published in NEJM.  Regardless of my opinion, it will undoubtedly be cited as additional evidence that long-term antibiotics don't alleviate long-term symptoms that stem from Lyme disease.

Edit: Corrected quotes in first paragraph.


Berende A, ter Hofstede HJ, Vos FJ, van Middendorp H, Vogelaar ML, Tromp M, van den Hoogen FH, Donders AR, Evers AW, & Kullberg BJ (2016). Randomized Trial of Longer-Term Therapy for Symptoms Attributed to Lyme Disease. The New England Journal of Medicine, 374 (13), 1209-20 PMID: 27028911

Melia MT, & Auwaerter PG (2016). Time for a Different Approach to Lyme Disease and Long-Term Symptoms. The New England Journal of Medicine, 374 (13), 1277-8 PMID: 27028918

Monday, March 21, 2016

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.


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

Thursday, February 11, 2016

How a new species of Lyme disease bacteria was discovered

A new agent of the tick-borne illness known as Lyme disease has emerged in the upper Midwest.  The bacterium is genetically related to Borrelia burgdorferi, until now believed to be the only cause of Lyme disease in the United States.  The name proposed for the bacterium is Borrelia mayonii because the work was conducted at the Mayo Clinic.  B. mayonii has not been detected in patients outside of the Midwest (so far).  The findings are described in The Lancet Infectious Diseases.

The new species was discovered at the Mayo Clinic during routine testing of specimens (blood, cerebral spinal fluid, and joint fluid) received from all regions of the U.S.  Over 100,000 specimens collected from 2003 through 2014 were tested for Lyme disease bacteria by real-time PCR . The PCR probes were designed to detect the oppA1 gene from Borrelia species belonging to the Lyme disease group, known in the scientific literature as "B. burgdorferi sensu lato."  The Lyme disease group comprises 18 species that fall into the same genetic cluster within the genus Borrelia.  They include species known or suspected to cause Lyme disease (B. burgdorferi, B. garinii, B. afzelii, B. spielmanii, B. valaisiana, B bissettii, B. bavariensis, and B. lusitaniae) and another ten species that do not cause illness.  The PCR probes do not react with DNA from species belonging to the other cluster of Borrelia, the relapsing fever group.

The key to the discovery of the new species was the melting temperature analysis routinely programmed onto the end of real-time PCR runs.  The oppA1 PCR products amplified from B. burgdorferi strains have melting temperatures of 63.6 through 64.9°C.  For other Lyme disease species, the melting temperature ranges from 52.3°C (B. valaisiana) to 59.2°C (B. californiensis).  Therefore, the melting temperature of the oppA1 PCR product was used to distinguish B. burgdorferi from other Lyme disease Borrelia.

Over 9,000 specimens were collected from the states of Minnesota, Wisconsin, and North Dakota from January 2012 through September 2014.  102 were PCR positive, and most of the PCR products had the melting temperature profile of B. burgdorferi.  However, six had melting temperatures ranging from 60.4°C to 61.2°C, too low to be B. burgdorferi but too high to be any other member of the Lyme disease group.  The novel spirochetes were cultured from the blood of two of the patients.  The DNA sequence of several "housekeeping" genes of the new isolates differed enough from those of other Borrelia species to signify that a new Borrelia species has been found.  The investigators named the new spirochete Borrelia mayonii.  No specimen collected from other regions of the U.S. exhibited the atypical melting temperatures, and neither did any collected earlier than 2012 from the Midwest.  These findings led the authors to conclude that B. mayonii has recently emerged in the upper Midwest and that the six patients are the first known cases of Lyme disease to be caused by the new species.

The investigators also collected Ixodes scapularis ticks in Wisconsin.  PCR and melting temperature analysis showed that 19 of 658 ticks (2.9%) were positive for B. mayonii, 195 (29.6%) positive for B. burgdorferi, and two positive for both.

One striking feature of B. mayonii infections is the large number of spirochetes circulating within the patients.  The densities ranged from 420,000 to 6,400,000 bacterial cells per milliliter, at least a hundred times higher than observed in the blood of patients with B. burgdorferi infections.  The numbers were high enough that spirochetes could be seen in blood collected from one of the patients.

Fig. 1b from Pritt et al., 2016

The six patients had many of the typical Lyme disease symptoms:  headache, neck pain, muscle aches, joint pain, and fatigue.  Although mild fever is also common in Lyme disease, two of the six patients had severe fevers with temperature readings approaching 40°C (104°F).  Four had nausea or were vomiting, which are also uncommon Lyme disease symptoms.  Two patients were hospitalized because of the severity of their illness.  Lyme disease may be missed in those infected with B. mayonii because of the unusual symptoms.

The standard two-tier antibody test, which uses B. burgdorferi antigens to detect reactive antibody, may help with the diagnosis.  Blood specimens from five of the six patients were tested.  Four patients either tested positive or, if negative initially, tested positive with blood drawn weeks later.  The one patient who tested negative had blood drawn only on the first day of illness, so it's likely that the antibody response hadn't kicked in fully.  The test appears to help with the diagnosis of Lyme disease caused by B. mayonii, but the number of patients tested was too small to draw firm conclusions.

The authors conclude:

In view of the differing clinical manifestations for patients infected with the novel B burgdorferi sensu lato genospecies, it is likely that Lyme borreliosis is not being considered—and therefore not diagnosed—in some patients with this infection. The clinical range of illness must be better defined in additional patients to ensure that physicians can recognise the infection and distinguish it from other tick-borne infections. Many tick-borne pathogens have global distribution, therefore studies are needed to establish the geographic distribution of human beings and ticks infected with the novel B. burgdorferi sensu lato genopecies. Finally, clinicians should be aware of the potential role of oppA1 PCR for diagnosing infection with this novel pathogen.


Pritt BS, Mead PS, Johnson DK, Neitzel DF, Respicio-Kingry LB, Davis JP, Schiffman E, Sloan LM, Schriefer ME, Replogle AJ, Paskewitz SM, Ray JA, Bjork J, Steward CR, Deedon A, Lee X, Kingry LC, Miller TK, Feist MA, Theel ES, Patel R, Irish CL, & Petersen JM (2016). Identification of a novel pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study. The Lancet. Infectious diseases. PMID: 26856777

Wednesday, November 11, 2015

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.


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

Sunday, September 6, 2015

Do Lyme disease spirochetes produce a toxin?

According to the current view of Lyme disease pathogenesis, tissue damage is caused by the inflammatory response to the spirochetes.  Borrelia species do not produce toxins that injure the host directly.  A new study published in BMC Microbiology may force us to modify our view.

The study shows that some Borrelia strains carry a set of genes with the potential to generate a peptide resembling streptolysin S (SLS), a potent toxin produced by the pathogen Streptococcus pyogenes.  The enzymes that produce SLS in S. pyogenes are expressed from a cluster of genes surrounding sagA, a tiny gene encoding the SLS precursor.  The peptide produced from sagA is nontoxic; it has to undergo several alterations to its structure to become toxic.  A critical modification is carried out by the SagBCD protein complex, which converts the side chains of cysteine, serine, and threonine into ring structures.
Figure 2 from Molloy et al., 2011

Other genes surrounding sagA encode a peptidase that is thought to trim the leader peptide from the amino terminus of the SLS precursor and an ABC transporter that may be responsible for expelling SLS from the cytoplasm.

Figure 1A from Molloy et al., 2011

SLS targets neutrophils and possibly other immune cells during S. pyogenes infection.  SLS-like toxins are also produced by other Gram-positive pathogens, including Staphylococcus aureus, Listeria monocytogenes and Clostridium botulinum.

The investigators mined the genomes of other bacteria in search for genes encoding the machinery that generates SLS-like toxins.  They found SLS-like gene clusters in various Firmicutes and Actinobacteria, both Gram-positive groups of bacteria.

The researchers also found the gene cluster in the genomes of Borrelia afzelii strain PKo, Borrelia valaisiana strain VS116, and Borrelia spielmanii strain A14S.  B. afzelii is a major cause of Lyme disease in Europe and Asia.  B. valaisiana and B. spielmanii are responsible for occasional cases of Lyme disease.

Figure 4 from Molloy et al., 2015.  Top: organization of SLS-like gene cluster in S. pyogenes and three Borrelia strains. Bottom: sequence of the SLS precursor (SagA) and the borrelial SLS-like precursors.

They also used PCR to screen the DNA of 140 patient and tick isolates of Lyme Borrelia for the genes encoding the SLS-like biosynthetic machinery.  Most of the isolates were obtained from Europe and the U.S., with a few coming from Asia.  Design of the PCR primers was based on the sequence of the B. valaisiana bvalB, bvalC, and bvalD genes, which encode homologs of the S. pyogenes sagB, sagC, and sagD gene products.  Most of the B. garinii, B. afzelii, B. valaisiana, B. spielmanii, and B. lusitaniae isolates that were examined tested positive.  On the other hand, none of the 22 isolates of B. burgdorferi or 13 isolates of B. bavariensis were PCR positive.  These results indicate that SLS-like sequences are widespread among Lyme disease spirochetes (though not in B. burgdorferi).

The next step was to show that the SLS-like borrelial gene actually encoded a peptide that damages mammalian cells.  A simple assay based on the ability of many toxins to rupture (hemolyze) red blood cell in vitro is available.  Hemolysis is measured easily by mixing the toxin with sheep red blood cells.  Hemoglobin released from the ruptured cells is quantified with a spectrophotometer.

They decided to test the SLS-like peptide encoded by B. valaisiana, BvalA, for hemolytic activity.  The researchers succeeded in expressing and purifying a recombinant form of BvalA.  Not surprisingly,  BvalA was not hemolytic because its amino acid side chains had to be converted into ring structures necessary for the peptide to injure red blood cells.  They wanted to mix BvalA with the BvalBCD protein complex so that the peptide would be modified, but they could not generate the protein complex.  Instead, they used the SagBCD complex from S. pyogenes to modify the BvalA peptide.  When they did this, they finally observed hemolytic activity.

Red blood cells are unlikely to be a major target of borrelial SLS-like peptides during infection.  So what is the real target?  More studies are needed to answer this question, but we should consider the possibility that the toxin has nothing to do with Lyme disease.  Instead, it may help the spirochete to survive during its residence within the tick vector.  A number of nonpathogenic bacteria carry gene clusters distantly related to the ones that produce SLS.  Several peptide toxins produced by these bacteria are known to kill competing microbes.  Like us humans, ticks have a microbiome inhabiting their gut.  Some Lyme spirochetes may need to secrete the toxin to ward off their microbial neighbors.


Molloy EM, Casjens SR, Cox CL, Maxson T, Ethridge NA, Margos G, Fingerle V, & Mitchell DA (2015). Identification of the minimal cytolytic unit for streptolysin S and an expansion of the toxin family. BMC Microbiology, 15 PMID: 26204951

Molloy EM, Cotter PD, Hill C, Mitchell DA, & Ross RP (2011). Streptolysin S-like virulence factors: the continuing sagA. Nature Reviews Microbiology, 9 (9), 670-81 PMID: 21822292