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1.  Precipitation of Iron on the Surface of Leptospira interrogans Is Associated with Mutation of the Stress Response Metalloprotease HtpX 
Applied and Environmental Microbiology  2013;79(15):4653-4660.
High concentrations of free metal ions in the environment can be detrimental to bacterial survival. However, bacteria utilize strategies, including the activation of stress response pathways and immobilizing chemical elements on their surface, to limit this toxicity. In this study, we characterized LA4131, the HtpX-like M48 metalloprotease from Leptospira interrogans, with a putative role in bacterial stress response and membrane homeostasis. Growth of the la4131 transposon mutant strain (L522) in 360 μM FeSO4 (10-fold the normal in vitro concentration) resulted in the production of an amorphous iron precipitate. Atomic force microscopy and transmission electron microscopy analysis of the strain demonstrated that precipitate production was associated with the generation and release of outer membrane vesicles (OMVs) from the leptospiral surface. Transcriptional studies indicated that inactivation of la4131 resulted in altered expression of a subset of metal toxicity and stress response genes. Combining these findings, this report describes OMV production in response to environmental stressors and associates OMV production with the in vitro activity of an HtpX-like metalloprotease.
doi:10.1128/AEM.01097-13
PMCID: PMC3719529  PMID: 23709510
2.  Colistin-Resistant, Lipopolysaccharide-Deficient Acinetobacter baumannii Responds to Lipopolysaccharide Loss through Increased Expression of Genes Involved in the Synthesis and Transport of Lipoproteins, Phospholipids, and Poly-β-1,6-N-Acetylglucosamine 
We recently demonstrated that colistin resistance in Acinetobacter baumannii can result from mutational inactivation of genes essential for lipid A biosynthesis (Moffatt JH, et al., Antimicrob. Agents Chemother. 54:4971–4977). Consequently, strains harboring these mutations are unable to produce the major Gram-negative bacterial surface component, lipopolysaccharide (LPS). To understand how A. baumannii compensates for the lack of LPS, we compared the transcriptional profile of the A. baumannii type strain ATCC 19606 to that of an isogenic, LPS-deficient, lpxA mutant strain. The analysis of the expression profiles indicated that the LPS-deficient strain showed increased expression of many genes involved in cell envelope and membrane biogenesis. In particular, upregulated genes included those involved in the Lol lipoprotein transport system and the Mla-retrograde phospholipid transport system. In addition, genes involved in the synthesis and transport of poly-β-1,6-N-acetylglucosamine (PNAG) also were upregulated, and a corresponding increase in PNAG production was observed. The LPS-deficient strain also exhibited the reduced expression of genes predicted to encode the fimbrial subunit FimA and a type VI secretion system (T6SS). The reduced expression of genes involved in T6SS correlated with the detection of the T6SS-effector protein AssC in culture supernatants of the A. baumannii wild-type strain but not in the LPS-deficient strain. Taken together, these data show that, in response to total LPS loss, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface.
doi:10.1128/AAC.05191-11
PMCID: PMC3256090  PMID: 22024825
3.  Colistin Resistance in Acinetobacter baumannii Is Mediated by Complete Loss of Lipopolysaccharide Production ▿  
Antimicrobial Agents and Chemotherapy  2010;54(12):4971-4977.
Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.
doi:10.1128/AAC.00834-10
PMCID: PMC2981238  PMID: 20855724
4.  Major Surface Protein LipL32 Is Not Required for Either Acute or Chronic Infection with Leptospira interrogans▿ †  
Infection and Immunity  2008;77(3):952-958.
Leptospira interrogans is responsible for leptospirosis, a zoonosis of worldwide distribution. LipL32 is the major outer membrane protein of pathogenic leptospires, accounting for up to 75% of total outer membrane protein. In recent times LipL32 has become the focus of intense study because of its surface location, dominance in the host immune response, and conservation among pathogenic species. In this study, an lipL32 mutant was constructed in L. interrogans using transposon mutagenesis. The lipL32 mutant had normal morphology and growth rate compared to the wild type and was equally adherent to extracellular matrix. Protein composition of the cell membranes was found to be largely unaffected by the loss of LipL32, with no obvious compensatory increase in other proteins. Microarray studies found no obvious stress response or upregulation of genes that may compensate for the loss of LipL32 but did suggest an association between LipL32 and the synthesis of heme and vitamin B12. When hamsters were inoculated by systemic and mucosal routes, the mutant caused acute severe disease manifestations that were indistinguishable from wild-type L. interrogans infection. In the rat model of chronic infection, the LipL32 mutant colonized the renal tubules as efficiently as the wild-type strain. In conclusion, this study showed that LipL32 does not play a role in either the acute or chronic models of infection. Considering the abundance and conservation of LipL32 among all pathogenic Leptospira spp. and its absence in saprophytic Leptospira, this finding is remarkable. The role of this protein in leptospiral biology and pathogenesis thus remains elusive.
doi:10.1128/IAI.01370-08
PMCID: PMC2643616  PMID: 19103763
5.  Genome-Wide Transposon Mutagenesis in Pathogenic Leptospira Species▿ ‡  
Infection and Immunity  2008;77(2):810-816.
Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans.
doi:10.1128/IAI.01293-08
PMCID: PMC2632054  PMID: 19047402

Results 1-5 (5)