Identification of Specific and Universal Virulence Factors in
            <i>Burkholderia cenocepacia</i>
            Strains by Using Multiple Infection Hosts

Uehlinger, Susanne; Schwager, Stephan; Bernier, Steve P.; Riedel, Katharina; Nguyen, D.T.; Sokol, Pamela A.; Eberl, Leo

doi:10.1128/iai.00398-09

Cited by 99 publications

(140 citation statements)

References 76 publications

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“…In only a few cases, however, have findings obtained in these models been validated using vertebrate hosts (mice) (2)(3)(4)(5)(6)(7)(8)(9)(10). A common caveat of these models is that the optimal temperature for maintaining them is below 28°C, whereas the optimum temperature for most human pathogens is 37°C.…”

Section: Drosophila Melanogaster Caenorhabditis Elegans and The Fishmentioning

confidence: 99%

Modeling Klebsiella pneumoniae Pathogenesis by Infection of the Wax Moth Galleria mellonella

et al. 2013

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The implementation of infection models that approximate human disease is essential for understanding pathogenesis at the molecular level and for testing new therapies before they are entered into clinical stages. Insects are increasingly being used as surrogate hosts because they share, with mammals, essential aspects of the innate immune response to infections. We examined whether the larva of the wax moth Galleria mellonella could be used as a host model to conceptually approximate Klebsiella pneumoniae-triggered pneumonia. We report that the G. mellonella model is capable of distinguishing between pathogenic and nonpathogenic Klebsiella strains. Moreover, K. pneumoniae infection of G. mellonella models some of the known features of Klebsiella-induced pneumonia, i.e., cell death associated with bacterial replication, avoidance of phagocytosis by phagocytes, and the attenuation of host defense responses, chiefly the production of antimicrobial factors. Similar to the case for the mouse pneumonia model, activation of innate responses improved G. mellonella survival against subsequent Klebsiella challenge. Virulence factors necessary in the mouse pneumonia model were also implicated in the Galleria model. We found that mutants lacking capsule polysaccharide, lipid A decorations, or the outer membrane proteins OmpA and OmpK36 were attenuated in Galleria. All mutants activated G. mellonella defensive responses. The Galleria model also allowed us to monitor Klebsiella gene expression. The expression levels of cps and the loci implicated in lipid A remodeling peaked during the first hours postinfection, in a PhoPQ-and PmrAB-governed process. Taken together, these results support the utility of G. mellonella as a surrogate host for assessing infections with K. pneumoniae. In 1890, Robert Koch formulated Koch's postulates as general guidelines for identifying disease-causing organisms. One century later, Stanley Falkow established the molecular version of Koch's postulates, this time to guide the identification of microbial genes encoding virulence factors. One of the key points of the molecular postulates is to test the virulence of a microorganism with an inactivated candidate virulence gene in an appropriate animal model. Therefore, the use of animal models to identify the virulence factors of human pathogens is indispensable. Currently, identification and characterization of novel virulence factors rely largely on assessing mutant bacteria for growth in the organs of infected mice. The dependence on mouse infection models limits large-scale analysis of virulence due to the large number of animals needed to obtain statistically significant results.To circumvent these issues, the search for alternative host models is ongoing. Ideally, these alternative models should be easy to maintain and infect, should be amenable to genetic manipulation, and should model aspects of vertebrate defenses upon infection, chiefly the immune response. The immune defense consists of two main parts, an innate and an adaptive response, wit...

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Section: Drosophila Melanogaster Caenorhabditis Elegans and The Fishmentioning

confidence: 99%

Modeling Klebsiella pneumoniae Pathogenesis by Infection of the Wax Moth Galleria mellonella

et al. 2013

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“…Also, B. cenocepacia can be transmitted from patient to patient (Drevinek & Mahenthiralingam, 2010). B. cenocepacia is pathogenic in several plant and non-mammalian animal infection models (Khodai-Kalaki et al, 2015;Thomson & Dennis, 2013;Uehlinger et al, 2009;Vergunst et al, 2010) and can survive intracellularly within epithelial cells (Burns et al, 1996;Sajjan et al, 2006), macrophages (Lamothe et al, 2007;Martin & Mohr, 2000;Saini et al, 1999) and amoebae (Lamothe et al, 2004;Marolda et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Quantification of type VI secretion system activity in macrophages infected with Burkholderia cenocepacia

Aubert

Valvano

2015

Microbiology

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The Gram-negative bacterial type VI secretion system (T6SS) delivers toxins to kill or inhibit the growth of susceptible bacteria, while other secretion systems target eukaryotic cells. Deletion of atsR, a negative regulator of virulence factors in B. cenocepacia K56-2, increases T6SS activity. Macrophages infected with a K56-2 DatsR mutant display dramatic alterations in their actin cytoskeleton architecture that rely on the T6SS, which is responsible for the inactivation of multiple Rho-family GTPases by an unknown mechanism. We employed a strategy to standardize the bacterial infection of macrophages and densitometrically quantify the T6SS-associated cellular phenotype, which allowed us to characterize the phenotype of systematic deletions of each gene within the T6SS cluster and ten vgrG genes in K56-2 DatsR. None of the genes from the T6SS core cluster nor the individual vgrG genes were directly responsible for the cytoskeletal changes in infected cells. However, a mutant strain with all vgrG genes deleted was unable to cause macrophage alterations. Despite not being able to identify a specific effector protein responsible for the cytoskeletal defects in macrophages, our strategy resulted in the identification of the critical core components and accessory proteins of the T6SS assembly machinery and provides a screening method to detect T6SS effectors targeting the actin cytoskeleton in macrophages by random mutagenesis.

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“…A putative novel transcription factor, sodium/hydrogen exchanger, gspJ (T2SS), and three metabolic genes were determined to be required for rapid pathogenicity to C. elegans, and five of these were also required for multihost pathogenicity. However, some virulence factors, such as those encoded by bnvR identified in the present study and those encoding a nematode toxin, aidA, the outer membrane protein opcI, and a component of T3SS, hldA, appear to be host specific (71). We further illustrate here how much remains to be learned about the complex problem of how select environmental microbes function as opportunistic pathogens.…”

Section: Vol 78 2010mentioning

confidence: 86%

The Type 2 Secretion Pseudopilin, gspJ , Is Required for Multihost Pathogenicity of Burkholderia cenocepacia AU1054

et al. 2010

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Burkholderia cenocepacia AU1054 is an opportunistic pathogen isolated from the blood of a person with cystic fibrosis. AU1054 is a multihost pathogen causing rapid pathogenicity to Caenorhabditis elegans nematodes. Within 24 h, AU1054 causes greater than 50% mortality, reduced growth, emaciated body, distended intestinal lumen, rectal swelling, and prolific infection of the nematode intestine. To determine virulence mechanisms, 3,000 transposon mutants were screened for attenuated virulence in nematodes. Fourteen virulence-attenuated mutants were isolated, and the mutant genes were identified. These genes included paaA, previously identified as being required for full virulence of B. cenocepacia K56-2. Six mutants were restored in virulence by complementation with their respective wild-type gene. One of these contained an insertion in gspJ, predicted to encode a pseudopilin component of the type 2 secretion system (T2SS). Nematodes infected with AU1054 gspJ had fewer bacteria present in the intestine than those infected with the wild type but still showed rectal swelling. The gspJ mutant was also defective in pathogenicity to onion and in degradation of polygalacturonic acid and casein. This result differs from previous studies where no or little role was found for T2SS in Burkholderia virulence, although virulence factors such as zinc metalloproteases and polygalacturonase are known to be secreted by the T2SS. This study highlights strain specific differences in B. cenocepacia virulence mechanisms important for understanding what enables environmental microbes to function as opportunistic pathogens.

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Identification of Specific and Universal Virulence Factors in Burkholderia cenocepacia Strains by Using Multiple Infection Hosts

Cited by 99 publications

References 76 publications

Modeling Klebsiella pneumoniae Pathogenesis by Infection of the Wax Moth Galleria mellonella

Modeling Klebsiella pneumoniae Pathogenesis by Infection of the Wax Moth Galleria mellonella

Quantification of type VI secretion system activity in macrophages infected with Burkholderia cenocepacia

The Type 2 Secretion Pseudopilin, gspJ , Is Required for Multihost Pathogenicity of Burkholderia cenocepacia AU1054

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