Identification of the lipopolysaccharide core of Yersinia pestis and Yersinia pseudotuberculosis as the receptor for bacteriophage φA1122

fA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. fA1122 infects Y. pestis grown both at 20 °C and at 37 °C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37 °C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30 °C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also fA1122 sensitive when grown at 22 °C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous fA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (L-glycero-D-manno-heptose/D-glucopyranose)-Kdo/Ko (3-deoxy-D-manno-oct-2-ulopyranosonic acid/D-glycero-D-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully fA1122 resistant. Our data thus conclusively demonstrated that the fA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis.

The pmrF polymyxin-resistance operon of Yersinia pseudotuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB, and is not required for virulence

The Yersinia pseudotuberculosis chromosome contains a seven-gene polycistronic unit (the pmrF operon) whose products share extensive homologies with their pmrF counterparts in Salmonella enterica serovar Typhimurium (S. typhimurium), another Gram-negative bacterial enteropathogen. This gene cluster is essential for addition of 4-aminoarabinose to the lipid moiety of LPS, as demonstrated by MALDI-TOF mass spectrometry of lipid A from both wild-type and pmrF-mutated strains. As in S. typhimurium, 4-aminoarabinose substitution of lipid A contributes to in vitro resistance of Y. pseudotuberculosis to the antimicrobial peptide polymyxin B. Whereas pmrF expression in S. typhimurium is mediated by both the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems, it appears to be PmrA-PmrB-independent in Y. pseudotuberculosis, with the response regulator PhoP interacting directly with the pmrF operon promoter region. This result reveals that the ubiquitous PmrA-PmrB regulatory system controls different regulons in distinct bacterial species. In addition, pmrF inactivation in Y. pseudotuberculosis has no effect on bacterial virulence in the mouse, again in contrast to the situation in S. typhimurium. The marked differences in pmrF operon regulation in these two phylogenetically close bacterial species may be related to their dissimilar lifestyles.

Yersinia pseudotuberculosis and Yersinia pestis show increased outer membrane permeability to hydrophobic agents which correlates with lipopolysaccharide acyl-chain fluidity

The hydrophobic probe N-phenyl-1-naphthylamine accumulated less in non-pathogenic Yersinia spp. and non-pathogenic and pathogenic Yersinia enterocolitica than in Yersinia pseudotuberculosis or Yersinia pestis. This was largely due to differences in the activity of efflux systems, but also to differences in outer membrane permeability because uptake of the probe in KCN/arsenate-poisoned cells was slower in the former group than in Y. pseudotuberculosis and Y. pestis. The probe accumulation rate was higher in Y. pseudotuberculosis and Y. pestis grown at 37 degrees C than at 26 degrees C and was always highest in Y. pestis. These yersiniae had LPSs with shorter polysaccharides than Y. enterocolitica, particularly when grown at 37 degrees C. Gelliquid-crystalline phase transitions (Tc 28-31 degrees C) were observed in LPS aggregates of Y. enterocolitica grown at 26 and 37 degrees C, with no differences between non-pathogenic and pathogenic strains. Y. pseudotuberculosis and Y. pestis LPSs showed no phase transitions and, although the fluidity of LPSs of Y. pseudotuberculosis and Y. enterocolitica grown at 26 degrees C were close below the Tc of the latter, they were always in a more fluid state than Y. enterocolitica LPS. Comparison with previous studies of Salmonella choleraesuis subsp. choleraesuis serotype minnesota rough LPS showed that the increased fluidity and absence of transition of Y. pseudotuberculosis and Y. pestis LPSs cannot be explained by their shorter polysaccharides and suggested differences at the lipid A/core level. It is proposed that differences in LPS-LPS interactions and efflux activity explain the above observations and reflect the adaptation of Yersinia spp. to different habitats.

Yersinia pseudotuberculosis and Yersinia pestis are more resistant to bactericidal cationic peptides than Yersinia enterocolitica

The action of bactericidal polycationic peptides was compared in Yersinia spp. by testing peptide binding to live cells and changes in outer membrane (OM) morphology and permeability. Moreover, polycation interaction with LPS was studied by measuring the dependence of dansylcadaverine displacement and zeta potential on polycation concentration. When growth at 37 degrees C, Yersinia pestis and Yersinia pseudotuberculosis bound less polymyxin B (PMB) than pathogenic or non-pathogenic Yersinia enterocolitica, regardless of virulence plasmid expression. Y. pseudotuberculosis OMs were unharmed by PMB concentrations causing extensive OM blebbing in Y. enterocolitica. The permeability to lysozyme caused by PMB was greater in Y. enterocolitica than in Y. pseudotuberculosis or Y. pestis and differences increased at 37 degrees C. Similar observations were made with other polycations using a polymyxin/novobiocin permeability assay. With LPS of cells grown at 26 degrees C, polycation binding was highest for Y. pseudotuberculosis and lowest for Y. pestis, with Y. enterocolitica yielding intermediate results which were lower for pathogenic than for non-pathogenic strains. With LPS of cells grown at 37 degrees C, polycation binding remained unchanged for Y. pestis and pathogenic Y. enterocolitica, increased for non-pathogenic Y. enterocolitica and decreased for Y. pseudotuberculosis to Y. pestis levels. Polycation binding related in part to differences in charge density (zeta potential) of LPS aggregates, suggesting similar effects at bacterial surfaces. It is suggested that species and temperature differences in polycation resistance relate to infection route, invasiveness and intracellular multiplication of Yersinia spp.

Pathogenic Yersinia enterocolitica strains increase the outer membrane permeability in response to environmental stimuli by modulating lipopolysaccharide fluidity and lipid A structure

Pathogenic biotypes of Yersinia enterocolitica (serotypes O:3, O:8, O:9, and O:13), but not environmental biotypes (serotypes O:5, O:6, O:7,8, and O:7,8,13,19), increased their permeability to hydrophobic probes when they were grown at pH 5.5 or in EGTA-supplemented (Ca(2+)-restricted) media at 37 degrees C. A similar observation was also made when representative strains of serotypes O:8 and O:5 were tested after brief contact with human monocytes. The increase in permeability was independent of the virulence plasmid. The role of lipopolysaccharide (LPS) in this phenomenon was examined by using Y. enterocolitica serotype O:8. LPS aggregates of bacteria grown in acidic or EGTA-supplemented broth took up more N-phenylnaphthylamine than LPS aggregates of bacteria grown in standard broth and also showed a marked increase in acyl chain fluidity which correlated with permeability, as determined by measurements obtained in the presence of hydrophobic dyes. No significant changes in O-antigen polymerization were observed, but lipid A acylation changed depending on the growth conditions. In standard medium at 37 degrees C, there were hexa-, penta-, and tetraacyl lipid A forms, and the pentaacyl form was dominant. The amount of tetraacyl lipid A increased in EGTA-supplemented and acidic media, and hexaacyl lipid A almost disappeared under the latter conditions. Our results suggest that pathogenic Y. enterocolitica strains modulate lipid A acylation coordinately with expression of virulence proteins, thus reducing LPS packing and increasing outer membrane permeability. The changes in permeability, LPS acyl chain fluidity, and lipid A acylation in pathogenic Y. enterocolitica strains approximate the characteristics in Yersinia pseudotuberculosis and Yersinia pestis and suggest that there is a common outer membrane pattern associated with pathogenicity.

Deciphering the Acylation Pattern of Yersinia enterocolitica Lipid A

Pathogenic bacteria may modify their surface to evade the host innate immune response. Yersinia enterocolitica modulates its lipopolysaccharide (LPS) lipid A structure, and the key regulatory signal is temperature. At 21°C, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37°C, Y. enterocolitica expresses a tetra-acylated lipid A consistent with the 3'-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37°C. Western blot analyses indicate that LpxR exhibits latency at 21°C, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo(2)-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of lpxR is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of Y. enterocolitica virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the lpxR mutant grown at 21°C. Mechanistically, our data revealed that the expressions of flhDC and rovA, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV)-encoded virulence factors Yops and YadA were not affected in the lpxR mutant. Finally, we establish that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the reduced activation of the LPS receptor by a LpxR-dependent deacylated LPS.

Molecular basis of Yersinia enterocolitica temperature-dependent resistance to antimicrobial peptides

Antimicrobial peptides (APs) belong to the arsenal of weapons of the innate immune system against infections. In the case of gram-negative bacteria, APs interact with the anionic lipid A moiety of the lipopolysaccharide (LPS). In yersiniae most virulence factors are temperature regulated. Studies from our laboratory demonstrated that Yersinia enterocolitica is more susceptible to polymyxin B, a model AP, when grown at 37°C than at 22°C (J. A. Bengoechea, R. Díaz, and I. Moriyón, Infect. Immun. 64:4891-4899, 1996), and here we have extended this observation to other APs, not structurally related to polymyxin B. Mechanistically, we demonstrate that the lipid A modifications with aminoarabinose and palmitate are downregulated at 37°C and that they contribute to AP resistance together with the LPS O-polysaccharide. Bacterial loads of lipid A mutants in Peyer's patches, liver, and spleen of orogastrically infected mice were lower than those of the wild-type strain at 3 and 7 days postinfection. PhoPQ and PmrAB two-component systems govern the expression of the loci required to modify lipid A with aminoarabinose and palmitate, and their expressions are also temperature regulated. Our findings support the notion that the temperature-dependent regulation of loci controlling lipid A modifications could be explained by H-NS-dependent negative regulation alleviated by RovA. In turn, our data also demonstrate that PhoPQ and PmrAB regulate positively the expression of rovA, the effect of PhoPQ being more important. However, rovA expression reached wild-type levels in the phoPQ pmrAB mutant background, hence indicating the existence of an unknown regulatory network controlling rovA expression in this background.

Characterization of the six glycosyltransferases involved in the biosynthesis of Yersinia enterocolitica serotype O:3 lipopolysaccharide outer core

Yersinia enterocolitica (Ye) is a gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage phiR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage phiR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities.

Role of lipid A acylation in Yersinia enterocolitica virulence

Yersinia enterocolitica is an important human pathogen. Y. enterocolitica must adapt to the host environment, and temperature is an important cue regulating the expression of most Yersinia virulence factors. Here, we report that Y. enterocolitica 8081 serotype O:8 synthesized tetra-acylated lipid A at 37 degrees C but that hexa-acylated lipid A predominated at 21 degrees C. By mass spectrometry and genetic methods, we have shown that the Y. enterocolitica msbB, htrB, and lpxP homologues encode the acyltransferases responsible for the addition of C(12), C(14) and C(16:1), respectively, to lipid A. The expression levels of the acyltransferases were temperature regulated. Levels of expression of msbB and lpxP were higher at 21 degrees C than at 37 degrees C, whereas the level of expression of htrB was higher at 37 degrees C. At 21 degrees C, an lpxP mutant was the strain most susceptible to polymyxin B, whereas at 37 degrees C, an htrB mutant was the most susceptible. We present evidence that the lipid A acylation status affects the expression of Yersinia virulence factors. Thus, expression of flhDC, the flagellar master regulatory operon, was downregulated in msbB and lpxP mutants, with a concomitant decrease in motility. Expression of the phospholipase yplA was also downregulated in both mutants. inv expression was downregulated in msbB and htrB mutants, and consistent with this finding, invasion of HeLa cells was diminished. However, the expression of rovA, the positive regulator of inv, was not affected in the mutants. The levels of pYV-encoded virulence factors Yops and YadA in the acyltransferase mutants were not affected. Finally, we show that only the htrB mutant was attenuated in vivo.

Characterization and biological role of the O-polysaccharide gene cluster of Yersinia enterocolitica serotype O:9

Yersinia enterocolitica serotype O:9 is a gram-negative enteropathogen that infects animals and humans. The role of lipopolysaccharide (LPS) in Y. enterocolitica O:9 pathogenesis, however, remains unclear. The O:9 LPS consists of lipid A to which is linked the inner core oligosaccharide, serving as an attachment site for both the outer core (OC) hexasaccharide and the O-polysaccharide (OPS; a homopolymer of N-formylperosamine). In this work, we cloned the OPS gene cluster of O:9 and identified 12 genes organized into four operons upstream of the gnd gene. Ten genes were predicted to encode glycosyltransferases, the ATP-binding cassette polysaccharide translocators, or enzymes required for the biosynthesis of GDP-N-formylperosamine. The two remaining genes within the OPS gene cluster, galF and galU, were not ascribed a clear function in OPS biosynthesis; however, the latter gene appeared to be essential for O:9. The biological functions of O:9 OPS and OC were studied using isogenic mutants lacking one or both of these LPS parts. We showed that OPS and OC confer resistance to human complement and polymyxin B; the OPS effect on polymyxin B resistance could be observed only in the absence of OC.

Expression of the Yersinia enterocolitica pYV-encoded type III secretion system is modulated by lipopolysaccharide O-antigen status

We show that the expression of a Yersinia enterocolitica O:8 pYV-encoded type III secretion system was altered in a rough mutant (YeO8-R) due to elevated levels of FlhDC. H-NS might underlie flhDC upregulation in YeO8-R, and the data suggest a relationship between the absence of O antigen and the expression of H-NS.

Experimental pig yersiniosis to assess attenuation of Yersinia enterocolitica O:8 mutant strains

An experimental oral pig model was used to assess the pathogenic and immunogenic potential of Yersinia enterocolitica serotype O:8 wild-type strain 8081-L2 and its lipopolysaccharide (LPS) mutant derivatives: a spontaneous rough mutant 8081-R2, strain 8081-DeltawzzGB expressing O-antigen with uncontrolled chain lengths, and strain 8081-wbcEGB expressing semirough LPS with only one O-unit. Microbiological and immunological parameters of the infected pigs were followed from day 7 to 60 postinfection. The wild-type and all LPS mutant strains persisted in the lymphoid tissue of tonsils and small intestines, causing asymptomatic infection without any pathological changes. Although the pig is known as a reservoir of Yersiniae, a precise analysis of pathogenic and immunogenic parameters based on different in vitro tests (hematological response, killing ability of leukocytes and blood sera, antibody response, hydrogen peroxide production by macrophages, classical and alternative pathways of complement activation), revealed significant attenuation in the pathogenicity of the LPS mutant strains but not the loss of immunogenic potential. In comparison with the other strains, strain 8081-DeltawzzGB demonstrated more continuous leucocytosis with monocytosis, higher invasive potential, significant activation of hydrogen peroxide production by macrophages and an effective immunoglobulin G immune response accompanied by relevant histological immunomorphological rearrangements.

Lipopolysaccharide O antigen status of Yersinia enterocolitica O:8 is essential for virulence and absence of O antigen affects the expression of other Yersinia virulence factors

Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria. Although much attention has been given to the biological effects of its lipid A portion, a great body of evidence indicates that its O chain polysaccharide (O antigen) portion plays an important role in the bacterium-host interplay. In this work we have studied in-depth the role of the O antigen in Yersinia enterocolitica serotype O:8 pathogenesis. We made a detailed virulence analysis of three mutants having different O antigen phenotypes: (i) LPS with no O antigen (rough mutant); (ii) LPS with one O unit (semirough mutant) and (iii) LPS with random distribution of O antigen chain lengths. We demonstrated that these LPS O antigen mutants were attenuated in virulence regardless of the infection route used. Co-infection experiments revealed that the rough and semirough mutants were severely impaired in their ability to colonize the Peyer's patches and in contrast to the wild-type strain they did not colonize spleen and liver. The mutant with random distribution of O antigen chain lengths, however, survived better but started to be cleared from mouse organs after 8 days. As an explanation to this attenuation we present here evidence that other Yersinia virulence factors depend on the presence of O antigen for their proper function and/or expression. We demonstrated that in the rough mutant: (i) the YadA function but not its expression was altered; (ii) Ail was not expressed and (iii) inv expression was downregulated. On the other hand, expression of flhDC, the flagellar master regulatory operon, was upregulated in this mutant with a concomitant increase in the production of flagellins. Finally, expression of yplA, encoding for the Yersinia phospholipase A, was also upregulated accompanied by an increased flagellar type III secretion system mediated secretion of YplA to culture medium. Together these findings suggest that the absence of O antigen in the outer membrane of Yersinia either directly or indirectly, for example through a cellular or membrane stress, could act as a regulatory signal.