BB0562 is a nutritional virulence determinant with lipase activity important for Borrelia burgdorferi infection and survival in fatty acid deficient environments

Abstract

The Lyme disease spirochete Borrelia burgdorferi relies on uptake of essential nutrients from its host environments for survival and infection. Therefore, nutrient acquisition mechanisms constitute key virulence properties of the pathogen, yet these mechanisms remain largely unknown. In vivo expression technology applied to B. burgdorferi (BbIVET) during mammalian infection identified gene bb0562, which encodes a hypothetical protein comprised of a conserved domain of unknown function, DUF3996. DUF3996 is also found across adjacent encoded hypothetical proteins BB0563 and BB0564, suggesting the possibility that the three proteins could be functionally related. Deletion of bb0562, bb0563 and bb0564 individually and together demonstrated that bb0562 alone was important for optimal disseminated infection in immunocompetent and immunocompromised mice by needle inoculation and tick bite transmission. Moreover, bb0562 promoted spirochete survival during the blood dissemination phase of infection. Gene bb0562 was also found to be important for spirochete growth in low serum media and the growth defect of Δbb0562 B. burgdorferi was rescued with the addition of various long chain fatty acids, particularly oleic acid. In mammals, fatty acids are primarily stored in fat droplets in the form of triglycerides. Strikingly, addition of glyceryl trioleate, the triglyceride form of oleic acid, to the low serum media did not rescue the growth defect of the mutant, suggesting bb0562 may be important for the release of fatty acids from triglycerides. Therefore, we searched for and identified two canonical GXSXG lipase motifs within BB0562, despite the lack of homology to known bacterial lipases. Purified BB0562 demonstrated lipolytic activity dependent on the catalytic serine residues within the two motifs. In sum, we have established that bb0562 is a novel nutritional virulence determinant, encoding a lipase that contributes to fatty acid scavenge for spirochete survival in environments deficient in free fatty acids including the mammalian host.

Riboflavin salvage by Borrelia burgdorferi supports carbon metabolism and is essential for survival in the tick vector

“…Fractionation of B. burgdorferi lysate into soluble and membrane fractions was performed as previously described (Kuhn et al, 2021). Briefly, wild‐type B. burgdorferi was grown to a density of ~1 × 10 8 cells/ml in BSK‐II medium.…”

“…The fusion construct was verified by restriction digest and Sanger sequencing. The pET43.1b‐ glpD opt plasmid was moved to NiCo21 (DE3) E. coli (New England Biolabs) for protein induction and purification, as previously described (Kuhn et al, 2021) (Figure S2). The glycerol 3‐phosphate dehydrogenase activity of recombinant Nus‐GlpD and Nus alone in the presence and absence of 10 μM FAD was determined using the G3PDH colorimetric assay kit (Abcam).…”

“…Ticks were dried with Whatman paper and allowed to recover for 48 h. The plasmid contents of all B. burgdorferi clones was verified by PCR (Jewett, Lawrence, et al, 2007). Three days post‐infection by immersion a subset the larval cohorts were allowed to feed to repletion on groups of 4 naïve C3H/HeN mice (Envigo) per B. burgdorferi clone, as previously described (Kuhn et al, 2021).…”

“…Groups of 10–20 unfed larvae were pooled per B. burgdorferi clone and DNA was isolated using a NucleoSpin tissue kit (Clontech Laboratories) per the manufacturer's instructions. Spirochete densities were determined by qPCR as previously described (Kuhn et al, 2021). Seven days post feeding to repletion, the number of B. burgdorferi per fed larva was determined by plating of individual triturated ticks in solid medium for colony forming units.…”

Use of host lipids by the Lyme disease spirochete may lead to biomarkers

“…Further, the study compared PC uptake in different bacteria and revealed that such uptake was specific to Borrelia and likely acquired by random diffusion. Borrelia lipase (bb0562) contributes to fatty acid scavenging and Borrelia survival under conditions in which free fatty acids are limited (12), but a comprehensive understanding of uptake will require further work. Gwynne and co-authors also demonstrated that the ratio of environmental lipids found in the growth media directly correlated with the proportion of lipids found in the Borrelia membrane (4).…”