A Transposon Site Hybridization Screen IdentifiesgalUandwecBCas Important for Survival of Yersinia pestis in Murine Macrophages

Abstract

Yersinia pestis is able to survive and replicate within murine macrophages. However, the mechanism by which Y. pestis promotes its intracellular survival is not well understood. To identify genes that are important for Y. pestis survival in macrophages, a library comprised of ϳ31,500 Y. pestis KIM6؉ transposon insertion mutants (input pool) was subjected to negative selection in primary murine macrophages. Genes underrepresented in the output pool of surviving bacteria were identified by transposon site hybridization to DNA oligonucleotide microarrays. The screen identified several genes known to be important for survival of Y. pestis in macrophages, including phoPQ and members of the PhoPQ regulon (e.g., pmrF). In addition, genes predicated to encode a glucose-1-phosphate uridylyltransferase (galU), a UDP-N-acetylglucosamine 2-epimerase (wecB) and a UDP-N-acetyl-D-mannosamine dehydrogenase (wecC) were identified in the screen. Viable-count assays demonstrated that a KIM6؉ galU mutant and a KIM6؉ wecBC mutant were defective for survival in murine macrophages. The galU mutant was studied further because of its strong phenotype. The KIM6؉ galU mutant exhibited increased susceptibility to the antimicrobial peptides polymyxin B and cathelicidin-related antimicrobial peptide (CRAMP). Polyacrylamide gel electrophoresis demonstrated that the lipooligosaccharide (LOS) of the galU mutant migrated faster than the LOS of the parent KIM6؉, suggesting the core was truncated. In addition, the analysis of LOS isolated from the galU mutant by mass spectrometry showed that aminoarabinose modification of lipid A is absent. Therefore, addition of aminoarabinose to lipid A and complete LOS core (galU), as well as enterobacterial common antigen (wecB and wecC), is important for survival of Y. pestis in macrophages.

Characterization of the Polymyxin B Resistome of Pseudomonas aeruginosa

“…Significantly, D-glucose is the most abundant sugar in the outer core oligosaccharide of LPS (36,37). The involvement of galU in polymyxin resistance has already been observed in other species such as Campylobacter jejuni (38), Proteus mirabilis (39), and Yersinia pestis (40). The product of PA5000, WapR, is a rhamnosyltransferase implicated in the synthesis of the capped core oligosaccharide, which is covalently linked to long-chain O polysaccharides of LPS (41,42).…”

“…In the case of galU, previous studies with P. mirabilis (39) and Y. pestis (40) revealed an impact of the galU mutation on lipid A modification and polymyxin B susceptibility. The product of galU, UDP-glucose phosphorylase, catalyzes the biosynthesis of UDP-glucose (49), a precursor of aminoarabinose, so the most likely explanation for the loss of lipid A modification would be the inability of this mutant strain to synthesize aminoarabinose.…”

Modern Views on Molecular Mechanisms of Plague Pathogenesis

“…важную роль при этом игра-ет наличие в лпс модификации фосфатных групп 4-аминоарабинозой, что приводит к уменьшению связывания бактерий с положительно заряженны-ми пептидами [20]. для осуществления этой моди-фикации бактериям необходим белок GalU (ури-дилтрансфераза глюкозо-1-фосфата), отсутствие которой делает бактерии более чувствительными к антимикробным пептидам и менее способными вы-живать в макрофагах [19]. резистентность бактерий к действию антимикробных пептидов связана и с наличием в олигосахаридном коре лпс концевого N-ацетилглюкозамина [17].…”

The importance of the magnesium transporter MgtB for virulence of Yersinia pseudotuberculosis and Yersinia pestis

“…The pathogenic Yersinia are known to have an intracellular phase during infection, and the inability to survive interaction with macrophages correlates with a reduction in virulence (Klein et al, 2012). Therefore, we aimed to determine the ability of the DmgtB mutants to invade and survive within macrophages.…”