The role of NagC in Yersinia pestis and Yersinia pseudotuberculosis biofilm development and insect transmission

There are three species of human-pathogenic Yersiniae; two of which cause gastrointestinal disease (Yersinia pseudotuberculosis and Yersinia enterocolitica) and one which causes plague (Yersinia pestis), one of the deadliest diseases in human history. Whilst Y. pseudotuberculosis and Y. enterocolitica are transmitted faeco-orally, Y. pestis is transmitted by insects, classically the Oriental rat flea, Xenopsylla cheopis. All Yersiniae are capable of a method of bacterial messaging known as quorum sensing (QS), which allows individual cells to communicate in a density dependent manner using signalling molecules such as N-acyl-homoserine lactones (AHLs). QS regulates several virulence phenotypes, including the production of Yersinia virulence factors (YOPs), secreted by the type three secretion system (T3SS) into mammalian host cells, auto-aggregation and biofilm formation. Biofilms are aggregates of bacteria within a protective exopolysaccharide matrix and are especially important for Y. pestis, as transmission of plague relies on the biofilm-mediated blockage of the proventriculus within the digestive system of a flea. Understanding the mechanisms of biofilm development are therefore potentially important for developing methods to prevent or control plague transmission. Recent research has also suggested a more prominent role in the spread of plague for other insect vectors, such as body lice (Pediculus corporis). In the Yersiniae, the extracellular polymeric substance (EPS) matrix is composed primarily of the polysaccharide N-acetyl-D-glucosamine (poly-GlcNAc) and extracellular DNA. The production of GlcNAc but not poly-GlcNAc is known to be regulated by NagC, a DNA binding protein responsible for the repression of nagE-nagBACD (GlcNAc catabolic operon) and activation of glmUS (GlcNAc biosynthesis operon) in Escherichia coli, but there is little information on the NagC-dependent regulation of GlcNAc metabolism or on poly-GlcNAc biosynthesis via the hmsHFRS operon in the Yersiniae. This study set out to investigate the contribution of NagC in Y. pseudotuberculosis and Y. pestis to QS, poly-GlcNAc production and biofilm formation in vitro and in vivo on and within the established C. elegans and insect vectors. Artificially fed colonies of X. cheopis and P. corporis were investigated for use in experimental infection models. For both insect species, defibrinated human blood fed through a collagen membrane was the most successful combination for feeding, although problems with reproducible control of environmental conditions, such as humidity, prevented the insect colonies from becoming established. To investigate the contribution of NagC to QS, YOP production, auto-aggregation, poly-GlcNAc biosynthesis and biofilm formation, a ΔnagC mutant was constructed in Y. pestis and compared with a Y. pseudotuberculosis ΔnagC mutant as well as with the corresponding wild type strains. NagC did appear to influence AHL and YOP production in both Y. pseudotuberculosis and Y. pestis. NagC did not influence auto-aggregation in Y. pestis, although the rate of auto-aggregation was slower for the Y. pseudotuberculosis ΔnagC mutant. NagC was however required for the production of poly-GlcNAc (Y. pseudotuberculosis) and biofilm formation (both Y. pseudotuberculosis and Y. pestis). To determine whether NagC directly regulates poly-GlcNAc biosynthesis, the Y. pestis nagC was expressed in E. coli, the recombinant protein purified and subjected to electrophoretic mobility shift assays. The data obtained show that NagC binds to the promoter regions of the GlcNAc metabolic operons nagE-nagBACD and glmUS, as well as hmsHFRS in Y. pestis and hence directly regulates the production of poly-GlcNAc. Using in vitro (glass) and in vivo (nematode and insect) models, both Y. pestis and Y. pseudotuberculosis ΔnagC mutants were evaluated for biofilm formation. In addition, a ‘fake flea’ proventriculus model was developed to investigate attachment and biofilm formation on a chitin substrate. The Y. pseudotuberculosis ΔnagC mutant showed reduced biofilm formation on the chitin surface, compared with the parental strain. On the nematode C. elegans, the Y. pestis ΔnagC mutant produced substantially less biofilm formation than the parent. When Y. pseudotuberculosis was fed to P. corporis, the ΔnagC mutant showed an increased ability to clear the infection and increased survival rates compared with insects fed the parent strain. Taken together, these results indicate that NagC plays a central role in biofilm formation and in the ability of the Yersiniae to be transmitted by insect vectors