Regulation of Lipid A Modifications by
            <i>Salmonella typhimurium</i>
            Virulence Genes
            <i>phoP-phoQ</i>

Guo, Lin; Lim, Kheng B.; Gunn, John S.; Bainbridge, Brian W.; Darveau, Richard P.; Hackett, Murray; Miller, Samuel I.

doi:10.1126/science.276.5310.250

Cited by 525 publications

(552 citation statements)

References 32 publications

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“…Bartonella LPS, however, does not induce the production of tumor necrosis factor alpha (23) and has low endotoxic activity as measured by toll-like receptor 2 signaling (41). This atypical host response to Bartonella LPS is most probably due to an unusual composition of the lipid A and attached long-chain fatty acids (11,14,41). The altered LPS of Bartonella may be responsible for the pathogen's ability to establish persistent bacteremic infections without causing significant clinical pathology within the host.…”

Section: Discussionmentioning

confidence: 99%

Predominant Outer Membrane Antigens of Bartonella henselae

Chenoweth

Greene

Krause³

et al. 2004

Infect Immun

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A hallmark of Bartonella henselae is persistent bacteremia in cats despite the presence of a vigorous host immune response. To understand better the long-term survival of B. henselae in cats, we examined the feline humoral immune response to B. henselae outer membrane (OM) proteins in naturally and experimentally infected cats. Initially, a panel of sera (n ‫؍‬ 42) collected throughout North America from naturally infected cats was used to probe B. henselae total membranes to detect commonly recognized antigens. Twelve antigens reacted with sera from at least 85% of cats, and five were recognized by sera from all cats. To localize these antigens further, OMs were purified on discontinuous sucrose density step gradients. Each membrane fraction (OM, hybrid or inner membrane [IM]) contained less than 1% of the total malate dehydrogenase activity (soluble marker), indicating very little contamination by cytoplasmic proteins. FtsI, an integral IM cell division protein, was used to identify the low-density fraction ( ‫؍‬ 1.13 g/cm 3 ) as putative IM (<5% of the total FtsI localized to the high-density fraction) while lipopolysaccharide (LPS) and Pap31, a homolog of the Bartonella quintana heme-binding protein A (HbpA), defined the high-density fraction ( ‫؍‬ 1.20 g/cm 3 ) as putative OM. Additionally, little evidence of cross-contamination between the IM and OM was evident by two-dimensional gel electrophoresis. When purified OMs were probed with feline sera, antigenic proteins profiles were very similar to those observed with total membranes, indicating that many, but not all, of the immunoreactive proteins detected in the initial immunoblots were OM components. Interestingly, two-dimensional immunoblots indicated that B. henselae LPS and members of the Hbp family of proteins did not appear to stimulate an humoral response in any infected cats. Seven proteins were recognized by at least 70% of sera tested, but only three were recognized by all sera. Nanospray-tandem mass spectrometry was used to identify OM components, including the immunodominant OM proteins. Recognition of the nonimmunogenic nature of the major OM components, such as LPS, and identification of the predominant immunogens should elucidate the mechanisms by which B. henselae establishes persistent bacteremic infections within cats. Additionally, the common antigens may serve as potential feline vaccine candidates to eliminate the pathogen from its animal reservoir.

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Section: Discussionmentioning

confidence: 99%

Predominant Outer Membrane Antigens of Bartonella henselae

Chenoweth

Greene

Krause³

et al. 2004

Infect Immun

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“…by down-regulating flagellin expression (Winter et al, 2008) or modifying LPS (Guo et al, 1997;Matsuura et al, 2012) of the commensal microbiota that often lack the capability to utilize these compounds (Thiennimitr et al, 2012). S. Typhimurium appears to be an ideal choice as a model organism for the establishment of a novel host-pathogen transcriptomic approach.…”

Section: Salmonella As a Model Organism For Host-pathogen Interactionmentioning

confidence: 99%

Dual RNA-seq of pathogen and host

2012

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SummaryThe infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections.However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell's physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies.The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed "Dual RNA-seq". Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella.Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and

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“…Some of the PhoP-induced enzymes promote increased bacterial resistance to antimicrobial peptides, a key component of host's innate immunity [10,17,21,22]. For example, PhoP-activated LPS modifications, including the addition of aminoarabinose and palmitate to lipid A, promote resistance to the antibiotic polymyxin and other cationic antimicrobial peptides [10,21,23]. Consistent with the addition of aminoarabinose to LPS, homologues of S. enterica enzymes necessary for aminoarabinose addition and resistance to polymyxin (PA3552-3554) were among the most highly induced proteins in this study (Table 2).…”

Section: Quantitative Proteomic Analysis Defined P Aeruginosa Virulementioning

confidence: 99%

“…Significant Gram-negative envelope remodeling is part of bacterial adaptation to magnesium limitation [10,23,11]. To define proteins that contribute to the envelope remodeling either structurally or by providing an enzymatic function to this process by mediating LPS modifications, total membrane protein of P. aeruginosa was also analyzed by quantitative proteomic analysis.…”

Section: Proteomic Analysis Revealed That Twenty P Aeruginosa Proteimentioning

confidence: 99%

Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation

Guina

Miller

et al. 2003

J. Am. Soc. Mass Spectrom.

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In this study, large-scale qualitative and quantitative proteomic technology was applied to the analysis of the opportunistic bacterial pathogen Pseudomonas aeruginosa grown under magnesium limitation, an environmental condition previously shown to induce expression of various virulence factors. For quantitative analysis, whole cell and membrane proteins were differentially labeled with isotope-coded affinity tag (ICAT) reagents and ICAT reagent-labeled peptides were separated by two-dimensional chromatography prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in an ion trap mass spectrometer (ITMS). To increase the number of protein identifications, gas-phase fractionation (GPF) in the m/z dimension was employed for analysis of ICAT peptides derived from whole cell extracts. The experiments confirmed expression of 1331 P. aeruginosa proteins of which 145 were differentially expressed upon limitation of magnesium. A number of conserved Gram-negative magnesium stress-response proteins involved in bacterial virulence were among the most abundant proteins induced in low magnesium. Comparative ICAT analysis of membrane versus whole cell protein indicated that growth of P. aeruginosa in low magnesium resulted in altered subcellular compartmentalization of large enzyme complexes such as ribosomes. This result was confirmed by 2-D PAGE analysis of P. aeruginosa outer membrane proteins. This study shows that large-scale quantitative proteomic technology can be successfully applied to the analysis of whole bacteria and to the discovery of functionally relevant biologic phenotypes.

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Regulation of Lipid A Modifications by Salmonella typhimurium Virulence Genes phoP-phoQ

Cited by 525 publications

References 32 publications

Predominant Outer Membrane Antigens of Bartonella henselae

Predominant Outer Membrane Antigens of Bartonella henselae

Dual RNA-seq of pathogen and host

Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation

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