Functional genome analysis of the intracellular lifestyle of Pseudomonas aeruginosa in phagocytes

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A VirB4 ATPase of the mobile accessory genome orchestrates core genome-encoded features of physiology, metabolism, and virulence of Pseudomonas aeruginosa TBCF10839

Pseudomonas aeruginosa TBCF10839 is a highly virulent strain that can persist and replicate in human neutrophils. Screening of a signature-tagged mutagenesis (STM) TBCF10839 transposon library in phagocytosis tests identified a mutant that carried the transposon in the VirB4 homolog 5PG21 of an integrative and conjugative element (ICE)-associated type IV secretion system of the pKLC102 subtype. 5P21 TBCF10839 insertion mutants were deficient in metabolic versatility, secretion, quorum sensing, and virulence. The mutants were efficiently killed in phagocytosis tests in vitro and were avirulent in an acute murine airway infection model in vivo. The inactivation of 5PG21 silenced the rhl, las, and pqs operons and the gene expression for the synthesis of hydrogen cyanide, the antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid, and the H2- and H3-type VI secretion systems and their associated effectors. The mutants were impaired in the utilization of carbon sources and stored compounds that are not funneled into intermediary metabolism. This showcase demonstrates that a single gene of the mobile accessory genome can become an essential element to operate the core genome-encoded features of metabolism and virulence

Identification and Characterization of a Novel Bacterial Virulence Factor That Shares Homology with Mammalian Toll/Interleukin-1 Receptor Family Proteins

Many important bacterial virulence factors act as mimics of mammalian proteins to subvert normal host cell processes. To identify bacterial protein mimics of components of the innate immune signaling pathway, we searched the bacterial genome database for proteins with homology to the Toll/interleukin-1 receptor (TIR) domain of the mammalian Toll-like receptors (TLRs) and their adaptor proteins. A previously uncharacterized gene, which we have named tlpA (for TIR-like protein A), was identified in the Salmonella enterica serovar Enteritidis genome that is predicted to encode a protein resembling mammalian TIR domains, We show that overexpression of TlpA in mammalian cells suppresses the ability of mammalian TIR-containing proteins TLR4, IL-1 receptor, and MyD88 to induce the transactivation and DNA-binding activities of NF-κB, a downstream target of the TIR signaling pathway. In addition, TlpA mimics the previously characterized Salmonella virulence factor SipB in its ability to induce activation of caspase-1 in a mammalian cell transfection model. Disruption of the chromosomal tlpA gene rendered a virulent serovar Enteritidis strain defective in intracellular survival and IL-1β secretion in a cell culture infection model using human THP1 macrophages. Bacteria with disrupted tlpA also displayed reduced lethality in mice, further confirming an important role for this factor in pathogenesis. Taken together, our findings demonstrate that the bacterial TIR-like protein TlpA is a novel prokaryotic modulator of NF-κB activity and IL-1β secretion that contributes to serovar Enteritidis virulence