Braun Lipoprotein (Lpp) Contributes to Virulence of Yersiniae: Potential Role of Lpp in Inducing Bubonic and Pneumonic Plague

Yersiniae are gram-negative bacilli in the family Enterobacteriaceae, and three species in the genus Yersinia are human pathogens. Yersinia pseudotuberculosis and Y. enterocolitica cause enteropathogenic infections that are typically self-limiting and are characterized by diarrhea, fever, and abdominal pain (52). Y. pestis is transmitted to humans through the bite of an infected flea or inhalation of the organisms, resulting in bubonic, pneumonic, or septicemic forms of plague. Y. pestis is historically credited for 200 million deaths worldwide and is also a potential agent of biowarfare or bioterrorism (18, 31, 34). The pathogenesis of yersinia infections is complex and multifactorial. A number of virulence-associated systems (mechanisms) are common among the three human-pathogenic yersiniae. For example, the type three secretion system (T3SS), encoded on a 70-kb plasmid, exists in all three species, and this plasmid is designated pCD1 in Y. pestis, pYVe in Y. enterocolitica, and pYV in Y. pseudotuberculosis (30, 45). Through the T3SS, a variety of effector proteins called Yops (for Yersinia outer membrane proteins) are translocated into the host cytosol and are absolutely required for the virulence of the yersiniae (30). On the other hand, each Yersinia species possesses its own unique set of virulence factors (30, 45). In the two enteropathogenic species, the virulence determinants Yad (for Yersinia adherence protein, formerly designated YopA) and invasin protein (Inv) play crucial roles in bacterial colonization and invasion of the intestinal epithelium but are not functional in Y. pestis (45). In addition to plasmid pCD1, Y. pestis harbors another two plasmids that are absent in the enteropathogenic yersiniae. Plasmid pPCP1 encodes the virulence determinant plasminogen activator (Pla), while pMT1, or pFra, encodes capsular antigen F1 and the murine toxin (45). It has been shown that the deletion of the 102-kb pigmentation (pgm) locus from the chromosome of Y. pestis attenuates its virulence, while this pgm locus is usually silent in Y. pseudotuberculosis (30). Recently, a transcriptional regulator, RovA, was identified in all three Yersinia species. RovA regulates a variety of genes and is specifically required for the development of bubonic plague in mice (8). Interestingly, a new set of T3SS-dependent proteins has been identified in Y. pestis and includes insecticidal-like proteins that are thought to function as transmission factors, contributing to flea morbidity by promoting colonization of the midgut (26). Therefore, the continued investigation of new virulence factors and virulence-associated systems is warranted to fully elucidate the mechanisms of yersinia pathogenesis. The outer membrane of gram-negative bacteria is comprised of many different proteins that help maintain the structural integrity of the bacterial cell envelope. Some of these proteins are covalently modified by the addition of a lipid, N-acyl-S-diacylglyceryl cysteine, and such modified proteins are collectively known as lipoproteins (37). One particularly abundant lipoprotein, designated murein (or Braun) lipoprotein (Lpp), is associated with the outer membranes of bacteria within the family Enterobacteriaceae. Originally identified in Escherichia coli, Lpp links the murein (peptidoglycan) layer to the outer bacterial membrane (29). Lpp has previously been demonstrated to play a role in the host's immune response against infections with some gram-negative enteric pathogens, such as E. coli, Salmonella enterica serovar Typhimurium, and Y. enterocolitica (65). Our earlier studies indicated that Lpp (6.3 kDa) purified from E. coli and Y. enterocolitica not only synergized with lipopolysaccharide (LPS) to induce septic shock, but also evoked the production of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in both LPS-responsive and LPS-nonresponsive mice and in mouse peritoneal exudate macrophages, suggesting an alternative signaling mechanism for Lpp (66). In fact, a subsequent study showed that Lpp signals through Toll-like receptor 2 (TLR-2) and not TLR-4 (2), which LPS utilizes for cell signaling. In our most recent studies, we provided evidence that S. enterica serovar Typhimurium harbors two copies of the lipoprotein gene, lppA and lppB, separated by 82 bp on the chromosome (22). Both single and double lpp gene deletions resulted in attenuation of bacteria in terms of their motility, invasiveness, and cytokine/chemokine production, and such mutants were avirulent in mice when administered by systemic and oral routes compared to the wild-type (WT) bacterium (21, 22, 58). By searching the National Center for Biotechnology Information database, we identified a homolog of the lpp gene in the genomes of all three pathogenic yersiniae (10, 15, 44). However unlike in S. enterica serovar Typhimurium, only a single copy of the lpp gene is present on the chromosome in Yersinia species. Consequently, in this study, we generated lpp mutants of WT/parental yersiniae (Y. pseudotuberculosis YPIII and Y. pestis KIM/D27 [pgm locus minus] and Y. pestis CO92) and evaluated them in mouse models of infection. Blood chemistries and blood cell counts, as well as organ pathology, were examined in specimens from the pneumonic and bubonic plague mouse models after infection with WT and Δlpp mutant strains of Y. pestis. We also evaluated the abilities of WT and mutant yersiniae to disseminate to the distal organs after infection. Additionally, the intracellular survival of WT and mutant Y. pestis CO92 bacteria was assessed in murine macrophages. Our data provided evidence that lpp mutants of Y. pseudotuberculosis and Y. pestis were attenuated in mice, an effect that could be complemented. More importantly, deletion of the lpp gene from the Y. pestis KIM/D27 mutant further attenuated the strain, and immunization of mice with this mutant provided protection to animals against pneumonic plague invoked by intranasal (i.n.) inoculation of Y. pestis CO92. To our knowledge, this is the first systematic study illustrating the role of Lpp in the pathogenesis of yersinia infection.