Mutational analyses of PS biosynthesis in the human commensal Bacteroides fragilis
Item statusRestricted Access
Embargo end date06/07/2020
Roberts, David William
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The Gram-negative bacterium Bacteroides fragilis is an obligate anaerobe that resides in the lumen of the human gastro-intestinal tract and has a symbiotic relationship with the host. To competitively colonise the mammalian intestine B. fragilis presents eight antigenically distinct phase variable capsular PSs (PS A-H) in the extra-cellular interface, and many extra-cytoplasmic proteins are O-glycosylated. B. fragilis is also an opportunistic pathogen; if contamination of the sterile peritoneal cavity transpires the zwitterionic nature the of capsular PS A is able to stimulate the formation of an abscess, enabling the bacterial cell to evade the host immune response. If untreated a B. fragilis infection can cause bacteraemia leading to death. Phenotypic analyses of the LPS expressed by B. fragilis9343 by different laboratories have used different methods of extraction, resulting in different conclusions as to the nature of this molecule. Evidence in the literature suggests that synthesis of PSs associated with the B. fragilis micro-capsule follows a similar pathway to E. coli K-antigen LPS (K-antigen of Smooth-LPS), and are anchored to Lipid-A. To test the hypothesis that the B. fragilis micro-capsule PSs are anchored to lipid-A, the deletion of WaaL, the O-antigen ligase, should prevent its biosynthesis. A recent publication has highlighted a ‘universally conserved’ WaaL motif H[NSQ]X9GXX[GTY]. Using in silico tools, a highly similarly WaaL motif [HNLYVLLILKIGIAV] was revealed in the polypeptide sequence encoded by BF4298. This result was unexpected as BF4298 is provisionally annotated to encode a Wzx flippase required in the B. fragilis O-glycosylation of proteins pathway. Deletion of an internal region BF4298 followed a precise strategy to generate markerless deletion B. fragilis ΔBF4298 strain. ΔBF4298 exhibits a complex ‘phenotype’ including a) inhibited growth in liquid medium, b) membrane swelling at cell poles, c) cell autoaggregation, sometimes with hundreds of adherents, with many exhibiting protuberances, d) a large, dark staining, biological mass enveloping a very large single or a few small tightly packed cells; this structure appears to be a huge capsule that my support cell division, e) cell structure deformations, f) expanded balloon-like erections, only observed as a part of a large cell conglomeration, g) very long filaments, some with extensive protrusions. Importantly, detection of classical MC PS ‘halo’ was abolished using five anti-MC mAbs in IFM micrographs. To provide supporting evidence it was hypothesized that deleting a non-essential Lipid-A biosynthesis gene may prevent anchoring of any MC PSs to an incomplete ‘rough’ LPS, as this moiety would no longer be a substrate for WaaL O-antigen ligase. A E. coli LpxL Lipid-A acyltransferase homolog was identified to be encoded by BF3626 in the genome of B. fragilis9343. The deletion stain B. fragilis ΔBF3626 exhibits a complex ‘deep’ rough’ phenotype similar to ΔBF4298, including abrogated detection of the MC PS in IFMs probed with anti-MC mAbs. The physicochemical properties of a high molecular mass PS chain anchored to Lipid- A may require a gentler extraction method to prevent shearing and insoluble aggregate formation associated with raised temperatures. Deoxycholate (DOC) is an ionic biological detergent that readily binds and forms micelles with LPS at room temperature. A novel method was specifically developed in this work to use DOC to replace SDS in PAGE to successfully resolve B. fragilis LPS. The LPS profiles of B. fragilis9343 and ΔBF3626 were resolved to an unprecedented level of clarity, indicating that the MC PS are not anchored to Lipid-A in this ‘deep rough’ mutant.