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