Zinc transporters YbtX and ZnuABC are required for the virulence of Yersinia pestis in bubonic and pneumonic plague in mice.

A number of bacterial pathogens require the ZnuABC Zinc (Zn2+) transporter and/or a second Zn2+ transport system to overcome Zn2+ sequestration by mammalian hosts. Previously we have shown that in addition to ZnuABC, Yersinia pestis possesses a second Zn2+ transporter that involves components of the yersiniabactin (Ybt), siderophore-dependent iron transport system. Synthesis of the Ybt siderophore and YbtX, a member of the major facilitator superfamily, are both critical components of the second Zn2+ transport system. Here we demonstrate that a ybtX znu double mutant is essentially avirulent in mouse models of bubonic and pneumonic plague while a ybtX mutant retains high virulence in both plague models. While sequestration of host Zn is a key nutritional immunity factor, excess Zn appears to have a significant antimicrobial role in controlling intracellular bacterial survival. Here, we demonstrate that ZntA, a Zn2+ exporter, plays a role in resistance to Zn toxicity in vitro, but that a zntA zur double mutant retains high virulence in both pneumonic and bubonic plague models and survival in macrophages. We also confirm that Ybt does not directly bind Zn2+in vitro under the conditions tested. However, we detect a significant increase in Zn2+-binding ability of filtered supernatants from a Ybt+ strain compared to those from a strain unable to produce the siderophore, supporting our previously published data that Ybt biosynthetic genes are involved in the production of a secreted Zn-binding molecule (zincophore). Our data suggest that Ybt or a modified Ybt participate in or promote Zn-binding activity in culture supernatants and is involved in Zn acquisition in Y. pestis.

The Yersinia pestis†HmsCDE regulatory system is essential for blockage of the oriental rat flea (Xenopsylla cheopis), a classic plague vector.

The second messenger molecule cyclic diguanylate is essential for Yersinia pestis biofilm formation that is important for blockage-dependent plague transmission from fleas to mammals. Two diguanylate cyclases (DGCs) HmsT and Y3730 (HmsD) are responsible for biofilm formation in vitro and biofilm-dependent blockage in the oriental rat flea Xenopsylla cheopis respectively. Here, we have identified a tripartite signalling system encoded by the y3729-y3731 operon that is responsible for regulation of biofilm formation in different environments. We present genetic evidence that a putative inner membrane-anchored protein with a large periplasmic domain Y3729 (HmsC) inhibits HmsD DGC activity in vitro while an outer membrane Pal-like putative lipoprotein Y3731 (HmsE) counteracts HmsC to activate HmsD in the gut of X. cheopis. We propose that HmsE is a critical element in the transduction of environmental signal(s) required for HmsD-dependent biofilm formation.

The Yersinia pestis siderophore, yersiniabactin, and the ZnuABC system both contribute to zinc acquisition and the development of lethal septicaemic plague in mice.

Bacterial pathogens must overcome host sequestration of zinc (Zn(2+) ), an essential micronutrient, during the infectious disease process. While the mechanisms to acquire chelated Zn(2+) by bacteria are largely undefined, many pathogens rely upon the ZnuABC family of ABC transporters. Here we show that in Yersinia pestis, irp2, a gene encoding the synthetase (HMWP2) for the siderophore yersiniabactin (Ybt) is required for growth under Zn(2+) -deficient conditions in a strain lacking ZnuABC. Moreover, growth stimulation with exogenous, purified apo-Ybt provides evidence that Ybt may serve as a zincophore for Zn(2+) acquisition. Studies with the Zn(2+) -dependent transcriptional reporter znuA::lacZ indicate that the ability to synthesize Ybt affects the levels of intracellular Zn(2+) . However, the outer membrane receptor Psn and TonB as well as the inner membrane (IM) ABC transporter YbtPQ, which are required for Fe(3+) acquisition by Ybt, are not needed for Ybt-dependent Zn(2+) uptake. In contrast, the predicted IM protein YbtX, a member of the Major Facilitator Superfamily, was essential for Ybt-dependent Zn(2+) uptake. Finally, we show that the ZnuABC system and the Ybt synthetase HMWP2, presumably by Ybt synthesis, both contribute to the development of a lethal infection in a septicaemic plague mouse model.

Manganese transporters Yfe and MntH are Fur-regulated and important for the virulence of Yersinia pestis.

Yersinia pestis has a flea-mammal-flea transmission cycle, and is a zoonotic pathogen that causes the systemic diseases bubonic and septicaemic plague in rodents and humans, as well as pneumonic plague in humans and non-human primates. Bubonic and pneumonic plague are quite different diseases that result from different routes of infection. Manganese (Mn) acquisition is critical for the growth and pathogenesis of a number of bacteria. The Yfe/Sit and/or MntH systems are the two prominent Mn transporters in Gram-negative bacteria. Previously we showed that the Y. pestis Yfe system transports Fe and Mn. Here we demonstrate that a mutation in yfe or mntH did not significantly affect in vitro aerobic growth under Mn-deficient conditions. A yfe mntH double mutant did exhibit a moderate growth defect which was alleviated by supplementation with Mn. No short-term energy-dependent uptake of (54)Mn was observed in this double mutant. Like the yfeA promoter, the mntH promoter was repressed by both Mn and Fe via Fur. Sequences upstream of the Fur binding sequence in the yfeA promoter converted an iron-repressible promoter to one that is also repressed by Mn and Fe. To our knowledge, this is the first report identifying cis promoter elements needed to alter cation specificities involved in transcriptional repression. Finally, the Y. pestis yfe mntH double mutant had an ~133-fold loss of virulence in a mouse model of bubonic plague but no virulence loss in the pneumonic plague model. This suggests that Mn availability, bacterial Mn requirements or Mn transporters used by Y. pestis are different in the lungs (pneumonic plague) compared with systemic disease.

Systematic analysis of cyclic di-GMP signalling enzymes and their role in biofilm formation and virulence in Yersinia pestis.

Cyclic di-GMP (c-di-GMP) is a signalling molecule that governs the transition between planktonic and biofilm states. Previously, we showed that the diguanylate cyclase HmsT and the putative c-di-GMP phosphodiesterase HmsP inversely regulate biofilm formation through control of HmsHFRS-dependent poly-ß-1,6-N-acetylglucosamine synthesis. Here, we systematically examine the functionality of the genes encoding putative c-di-GMP metabolic enzymes in Yersinia pestis. We determine that, in addition to hmsT and hmsP, only the gene y3730 encodes a functional enzyme capable of synthesizing c-di-GMP. The seven remaining genes are pseudogenes or encode proteins that do not function catalytically or are not expressed. Furthermore, we show that HmsP has c-di-GMP-specific phosphodiesterase activity. We report that a mutant incapable of c-di-GMP synthesis is unaffected in virulence in plague mouse models. Conversely, an hmsP mutant, unable to degrade c-di-GMP, is defective in virulence by a subcutaneous route of infection due to poly-ß-1,6-N-acetylglucosamine overproduction. This suggests that c-di-GMP signalling is not only dispensable but deleterious for Y. pestis virulence. Our results show that a key event in the evolution of Y. pestis from the ancestral Yersinia pseudotuberculosis was a significant reduction in the complexity of its c-di-GMP signalling network likely resulting from the different disease cycles of these human pathogens.

The yersiniabactin transport system is critical for the pathogenesis of bubonic and pneumonic plague.

Iron acquisition from the host is an important step in the pathogenic process. While Yersinia pestis has multiple iron transporters, the yersiniabactin (Ybt) siderophore-dependent system plays a major role in iron acquisition in vitro and in vivo. In this study, we determined that the Ybt system is required for the use of iron bound by transferrin and lactoferrin and examined the importance of the Ybt system for virulence in mouse models of bubonic and pneumonic plague. Y. pestis mutants unable to either transport Ybt or synthesize the siderophore were both essentially avirulent via subcutaneous injection (bubonic plague model). Surprisingly, via intranasal instillation (pneumonic plague model), we saw a difference in the virulence of Ybt biosynthetic and transport mutants. Ybt biosynthetic mutants displayed an approximately 24-fold-higher 50% lethal dose (LD(50)) than transport mutants. In contrast, under iron-restricted conditions in vitro, a Ybt transport mutant had a more severe growth defect than the Ybt biosynthetic mutant. Finally, a Delta pgm mutant had a greater loss of virulence than the Ybt biosynthetic mutant, indicating that the 102-kb pgm locus encodes a virulence factor, in addition to Ybt, that plays a role in the pathogenesis of pneumonic plague.

Insights into Yersinia pestis biofilm development: topology and co-interaction of Hms inner membrane proteins involved in exopolysaccharide production.

Primarily, three operons, hmsHFRS, hmsT and hmsP, are responsible for the development of a Yersinia pestis biofilm, which is essential for blockage-dependent transmission of plague from fleas to mammals. Here, using specific antibodies, a polymeric beta-1,6-N-acetyl-d-glucosamine-like polysaccharide was detected in the extracellular matrix of hmsHFRS-dependent Y. pestis biofilm. The production of this exopolysaccharide (EPS) was controlled by diguanylate cyclase HmsT and EAL domain phosphodiesterase HmsP, acting as positive and negative regulators respectively. Cellular compartmentalization of soluble segments of Hms inner membrane proteins, including the putative glycosyltransferase domain of HmsR, the diguanylate cyclase/GGDEF domain of HmsT and the phosphodiesterase/EAL domain of HmsP, was determined by a combination of topology prediction algorithms and construction of C-terminal translational fusions with beta-galactosidase and alkaline phosphatase. Multiple interactions of Hms inner membrane proteins were detected using bacterial cAMP based two-hybrid system. Biochemical analyses confirmed some of these protein-protein interactions. Our results indicate that synthesis and regulation of the Y. pestis biofilm EPS occurs in the cytoplasm by a proposed Hms enzymatic complex.

Molecular characterization of L-413C, a P2-related plague diagnostic bacteriophage.

Our analysis of the plague diagnostic phage L-413C genome sequence and structure reveals that L-413C is highly similar and collinear with enterobacteriophage P2, though important differences were found. Of special interest was the mosaic nature of the tail fiber protein H in L-413C, given the differentiating specificity of this phage for Yersinia pestis vs. Yersinia pseudotuberculosis. While the N-terminal 207 and C-terminal 137 amino acids of L-413C display significant homology with the P2 H protein, a large (465 amino acid) middle section appears to be derived from a T4-related H protein, with highest similarity to the T6 and RB32 distal tail fibers. This finding along with appropriate preadsorption experiments suggest that the unique H protein of L-413C may be responsible for the specificity of this phage for Y. pestis, and that the Y. pestis receptors that are recognized and bound by L-413C either do not exist in Y. pseudotuberculosis or have a different structure.

Regulation of biofilm formation in Yersinia pestis.

Plague biofilm development is controlled by positive (HmsT) and negative (HmsP) regulators. The GGDEF-domain protein HmsT appears to have diguanylate cyclase activity to synthesize bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) from 2 GTP molecules. The EAL domain of HmsP has phosphodiesterase activity and likely degrades c-di-GMP. This second messenger molecule probably influences biofilm development by activating the glycosyl transferase activity of HmsR. Here we demonstrate the in vitro pH optimum for phosphodiesterase activity of HmsP and that an alanine substitution in residue L508, D626, or E686 within the EAL domain affects this enzymatic activity and the biological function of the protein. Finally, protein-protein interactions and the cytoplasmic location of the enzymatic domains of HmsT and HmsP are evaluated.

Identification of critical amino acid residues in the plague biofilm Hms proteins.

Yersinia pestis biofilm formation causes massive adsorption of haemin or Congo red in vitro as well as colonization and eventual blockage of the flea proventriculus in vivo. This blockage allows effective transmission of plague from some fleas, like the oriental rat flea, to mammals. Four Hms proteins, HmsH, HmsF, HmsR and HmsS, are essential for biofilm formation, with HmsT and HmsP acting as positive and negative regulators, respectively. HmsH has a beta-barrel structure with a large periplasmic domain while HmsF possesses polysaccharide deacetylase and COG1649 domains. HmsR is a putative glycosyltransferase while HmsS has no recognized domains. In this study, specific amino acids within conserved domains or within regions of high similarity in HmsH, HmsF, HmsR and HmsS proteins were selected for site-directed mutagenesis. Some but not all of the substitutions in HmsS and within the periplasmic domain of HmsH were critical for protein function. Substitutions within the glycosyltransferase domain of HmsR and the deacetylase domain of HmsF abolished biofilm formation in Y. pestis. Surprisingly, substitution of highly conserved residues within COG1649 did not affect HmsF function.

Hierarchy of iron uptake systems: Yfu and Yiu are functional in Yersinia pestis.

In addition to the yersiniabactin (Ybt) siderophore-dependent system, two inorganic iron ABC transport systems of Yersinia pestis, Yfe and Yfu, have been characterized. Here we show that the Yfu system functions in Y. pestis: a Ybt- Yfe- Yfu- mutant exhibited a greater growth defect under iron-deficient conditions than its Ybt- Yfe- parental strain. We also demonstrate that another putative Y. pestis iron uptake system, Yiu, which potentially encodes an outer membrane receptor, YiuR, and an ABC iron transport cassette, YiuABC, is functional. The cloned yiuABC operon restored growth of an enterobactin-deficient mutant Escherichia coli strain, 1017, under iron-chelated conditions. Iron uptake by the Yiu system in Y. pestis was demonstrated only when the Ybt, Yfe, and Yfu systems were mutated. Using a yiuA::lacZ fusion, we show that the yiuABC promoter is repressed by iron through Fur. A mouse model of bubonic plague failed to show a significant role for the Yiu system in the disease process. These results demonstrate that two additional iron transporters are functional in Y. pestis and indicate that there is a hierarchy of iron transporters, with Ybt being most effective and Yiu being the least effective of those systems which have been characterized.

The phosphodiesterase activity of the HmsP EAL domain is required for negative regulation of biofilm formation in Yersinia pestis.

In Yersinia pestis, biofilm formation is stimulated by HmsT, a GGDEF-domain containing protein that synthesizes cyclic-di-GMP (c-di-GMP), and inhibited by HmsP, an EAL-domain protein. Only the EAL-domain portion of HmsP is required to inhibit biofilm formation. The EAL domain of HmsP was purified as a 6XHis-tag fusion protein and demonstrated to have phosphodiesterase activity using bis(p-nitrophenyl) phosphate (bis-pNPP) as a substrate. This enzymatic activity was strictly manganese dependent. A critical residue (E506) of HmsP within the EAL domain, that is required for inhibition of biofilm formation, is also essential for this phosphodiesterase activity. While the proposed function of EAL-domain proteins is to linearize c-di-GMP, this is a direct demonstration of the required phosphodiesterase activity of a purified EAL-domain protein.

HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms-dependent biofilm formation in Yersinia pestis.

The Hms(+) phenotype of Yersinia pestis promotes the binding of haemin or Congo red (CR) to the cell surface at temperatures below 34 degrees C. We previously demonstrated that temperature regulation of the Hms(+) phenotype is not controlled at the level of transcription. Instead, HmsH, HmsR and HmsT are degraded upon a temperature shift from 26 degrees C to 37 degrees C. We used random transposon mutagenesis to identify new genes involved in the temperature-regulated expression of the Hms phenotype. One of these genes, which we designated hmsP, encodes a putative phosphodiesterase with a conserved EAL motif. Mutations in hmsP caused formation of red colonies on CR plates at 26 degrees C and 37 degrees C. Strains complemented with hmsP(+) on a plasmid form white colonies at both temperatures. We used a crystal violet assay and confocal laser scanning microscopy to demonstrate Hms-dependent biofilm formation by Y. pestis cells. Y. pestis Hms(+) strains grown at 26 degrees C but not at 37 degrees C form a biofilm on borosilicate glass surfaces. Strains that either overexpress HmsT (a GGDEF domain protein) or have a mutation in hmsP produced an extremely thick biofilm. Alanine substitutions for each of the GGEE residues (amino acids 296-299) of HmsT as well as the E506 and L508 residues of HmsP caused a loss of function. We propose that HmsT and HmsP together control the amount of biofilm produced in Y. pestis. Degradation of HmsT at 37 degrees C may be a critical factor in controlling the temperature-dependent expression of the Hms biofilm.

Temperature regulation of the hemin storage (Hms+) phenotype of Yersinia pestis is posttranscriptional.

In Yersinia pestis, the Congo red (and hemin) binding that is characteristic of the Hms+ phenotype occurs at temperatures up to 34 degrees C but not at higher temperatures. Manifestation of the Hms+ phenotype requires at least five proteins (HmsH, -F, -R, -S, and -T) that are organized into two separate operons: hmsHFRS and hmsT. HmsH and HmsF are outer membrane proteins, while HmsR, HmsS, and HmsT are predicted to be inner membrane proteins. We have used transcriptional reporter constructs, RNA dot blots, and Western blots to examine the expression of hms operons and proteins. Our studies indicate that transcription from the hmsHFRS and hmsT promoters is not regulated by the iron status of the cells, growth temperature, or any of the Hms proteins. In addition, the level of mRNA for both operons is not significantly affected by growth temperature. However, protein levels of HmsH, HmsR, and HmsT in cells grown at 37 degrees C are very low compared to those in cells grown at 26 degrees C, while the amounts of HmsF and HmsS show only a moderate reduction at the higher growth temperature. Neither the Pla protease nor a putative endopeptidase (Y2360) encoded upstream of hmsH is essential for temperature regulation of the Hms+ phenotype. However, HmsT at 37 degrees C is sensitive to degradation by Lon and/or ClpPX. Thus, the stability of HmsH, HmsR, and HmsT proteins likely plays a role in temperature regulation of the Hms+ phenotype of Y. pestis.