Structural characterization of lipo-oligosaccharide (LOS) from Yersinia pestis: Regulation of LOS structure by the PhoPQ system

Department of Biological Sciences, Wolfson Building, Imperial College, London, SW7 2AY, UK.
Molecular Microbiology (Impact Factor: 4.42). 07/2002; 44(6):1637-50. DOI: 10.1046/j.1365-2958.2002.02990.x
Source: PubMed


The two-component regulatory system PhoPQ has been shown to regulate the expression of virulence factors in a number of bacterial species. For one such virulence factor, lipopolysaccharide (LPS), the PhoPQ system has been shown to regulate structural modifications in Salmonella enterica var Typhimurium. In Yersinia pestis, which expresses lipo-oligosaccharide (LOS), a PhoPQ regulatory system has been identified and an isogenic mutant constructed. To investigate potential modifications to LOS from Y. pestis, which to date has not been fully characterized, purified LOS from wild-type plague and the phoP defective mutant were analysed by mass spectrometry. Here we report the structural characterization of LOS from Y. pestis and the direct comparison of LOS from a phoP mutant. Structural modifications to lipid A, the host signalling portion of LOS, were not detected but analysis of the core revealed the expression of two distinct molecular species in wild-type LOS, differing in terminal galactose or heptose. The phoP mutant was restricted to the expression of a single molecular species, containing terminal heptose. The minimum inhibitory concentration of cationic antimicrobial peptides for the two strains was determined and compared with the wild-type: the phoP mutant was highly sensitive to polymyxin. Thus, LOS modification is under the control of the PhoPQ regulatory system and the ability to alter LOS structure may be required for survival of Y. pestis within the mammalian and/or flea host.

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Available from: Howard Morris, Sep 16, 2015
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    • "Intracellular growth of Y. pestis in macrophages occurs at early stages of systemic infection [3]. A phoP null mutant of Y. pestis showed reduced ability to survive in macrophages and human neutrophils, as well as under in vitro conditions of low pH, oxidative stress, high osmolarity, and antimicrobial peptides [4], [5], [6]; this mutant is slightly attenuated in mice [4]. As a global regulator, PhoP controls a very complex regulatory cascade in Y. pestis [7], [8], [9]. "
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    ABSTRACT: Yersinia pestis is the causative agent of plague. The two transcriptional regulators, PhoP and RovA, are required for the virulence of Y. pestis through the regulation of various virulence-associated loci. They are the global regulators controlling two distinct large complexes of cellular pathways. Based on the LacZ fusion, primer extension, gel mobility shift, and DNase I footprinting assays, RovA is shown to recognize both of the two promoters of its gene in Y. pestis. The autoregulation of RovA appears to be a conserved mechanism shared by Y. pestis and its closely related progenitor, Y. pseudotuberculosis. In Y. pestis, the PhoP regulator responds to low magnesium signals and then negatively controls only one of the two promoters of rovA through PhoP-promoter DNA association. RovA is a direct transcriptional activator for its own gene in Y. pestis, while PhoP recognizes the promoter region of rovA to repress its transcription. The direct regulatory association between PhoP and RovA bridges the PhoP and RovA regulons in Y. pestis.
    Full-text · Article · Sep 2011 · PLoS ONE
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    • "Disruption of the phoP gene renders Y. pestis more sensitive to low pH, oxidative stress and antimicrobial peptides that are found to be killing mechanisms for invading pathogen by macrophage [8,15]. When the phoP gene of Y. pestis strain 201 used in this study was disrupted using a lambda Red recombination system [16], similar phenotypes of this phoP null mutant were also observed (data not shown). "
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    ABSTRACT: The transcription regulator PhoP has been shown to be important for Y. pestis survival in macrophages and under various in vitro stresses. However, the mechanism by which PhoP promotes bacterial intracellular survival is not fully understood. Our previous microarray analysis suggested that PhoP governed a wide set of cellular pathways in Y. pestis. A series of biochemical experiments were done herein to study members of the PhoP regulon of Y. pestis biovar Microtus. By using gel mobility shift assay and quantitative RT-PCR, a total of 30 putative transcription units were characterized as direct PhoP targets. The primer extension assay was further used to determine the transcription start sites of 18 PhoP-dependent promoters and to localize the -10 and -35 elements. The DNase I footprinting was used to identify the PhoP-binding sites within 17 PhoP-dependent promoters, enabling the identification of PhoP box and matrix that both represented the conserved signals for PhoP recognition in Y. pestis. Data presented here providing a good basis for modeling PhoP-promoter DNA interactions that is crucial to the PhoP-mediated transcriptional regulation. The proven direct PhoP targets include nine genes encoding regulators and 21 genes or operons with functions of detoxification, protection against DNA damages, resistance to antimicrobial peptides, and adaptation to magnesium limitation. We can presume that PhoP is a global regulator that controls a complex regulatory cascade by a mechanism of not only directly controlling the expression of specific genes, but also indirectly regulating various cellular pathways by acting on a set of dedicated regulators. These results help us gain insights into the PhoP-dependent mechanisms by which Y. pestis survives the antibacterial strategies employed by host macrophages.
    Full-text · Article · Feb 2008 · BMC Genomics
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    • "The variant was completely avirulent in mice at 10 8 organisms by the parenteral route. None of the absent plasmids (pFra, pCD, pPst) or missing parts of the genome (Dpgm) of A. P. Anisimov and others the mutant strains contained genes for LPS biogenesis, and did not exert influence on LPS structure when compared with the 'wild-type' in any of the strains (Hitchen et al., 2002; Kawahara et al., 2002; Knirel et al., 2005a; Rebeil et al., 2004) except for specially generated mutants (DlpxM mutants in this study). "
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    ABSTRACT: Yersinia pestis undergoes an obligate flea-rodent-flea enzootic life cycle. The rapidly fatal properties of Y. pestis are responsible for the organism's sustained survival in natural plague foci. Lipopolysaccharide (LPS) plays several roles in Y. pestis pathogenesis, prominent among them being resistance to host immune effectors and induction of a septic-shock state during the terminal phases of infection. LPS is acylated with 4-6 fatty acids, the number varying with growth temperature and affecting the molecule's toxic properties. Y. pestis mutants were constructed with a deletion insertion in the lpxM gene in both virulent and attenuated strains, preventing the organisms from synthesizing the most toxic hexa-acylated lipid A molecule when grown at 25 degrees C. The virulence and/or protective potency of pathogenic and attenuated Y. pestis DeltalpxM mutants were then examined in a mouse model. The DeltalpxM mutation in a virulent strain led to no change in the LD(50) value compared to that of the parental strain, while the DeltalpxM mutation in attenuated strains led to a modest 2.5-16-fold reduction in virulence. LPS preparations containing fully hexa-acylated lipid A were ten times more toxic in actinomycin D-treated mice then preparations lacking this lipid A isoform, although this was not significant (P>0.05). The DeltalpxM mutation in vaccine strain EV caused a significant increase in its protective potency. These studies suggest there is little impact from lipid A modifications on the virulence of Y. pestis strains but there are potential improvements in the protective properties in attenuated vaccine strains.
    Full-text · Article · May 2007 · Journal of Medical Microbiology
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