Identification of Conserved ABC Importers Necessary for Intracellular Survival of Legionella pneumophila in Multiple Hosts.

It is established that the human pathogen Legionella pneumophila becomes significantly augmented for infection of macrophages after intracellular growth in amoebae when compared to like-strains cultivated in laboratory media. Based on this observation, we reasoned that the most critical virulence determinants of L.p. are expressed by responding to stimuli generated by the protozoan host specifically; a process we term "protozoan-priming." We sought to identify L.p. virulence factors that were required for replication in amoebae in order to highlight the genes necessary for production of the most infectious form of the bacterium. Using a transposon mutagenesis screen, we successfully identified 12 insertions that produced bacteria severely attenuated for growth in amoebae, while retaining a functional Dot/Icm type IVb secretion system. Seven of these insertion mutants were found dispensable for growth in macrophages, revealing attractive therapeutic targets that reside upstream of the pathogen-human interface. Two candidates identified, lpg0730 and lpg0122 were required for survival and replication in amoebae and macrophage host cells. Both genes are conserved among numerous important human pathogenic bacteria that can persist or replicate in amoebae. Each gene encodes a component of an ATP binding cassette (ABC) transport complex of unknown function. We demonstrate the lpg0730 ortholog in Francisella tularensis subsp. novicida to be essential for colonization of both protozoan and mammalian host cells, highlighting conserved survival mechanisms employed by bacteria that utilize protozoa as an environmental reservoir for replication.

ChIP-Seq Analysis of the σE Regulon of Salmonella enterica Serovar Typhimurium Reveals New Genes Implicated in Heat Shock and Oxidative Stress Response.

The alternative sigma factor σE functions to maintain bacterial homeostasis and membrane integrity in response to extracytoplasmic stress by regulating thousands of genes both directly and indirectly. The transcriptional regulatory network governed by σE in Salmonella and E. coli has been examined using microarray, however a genome-wide analysis of σE-binding sites in Salmonella has not yet been reported. We infected macrophages with Salmonella Typhimurium over a select time course. Using chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq), 31 σE-binding sites were identified. Seventeen sites were new, which included outer membrane proteins, a quorum-sensing protein, a cell division factor, and a signal transduction modulator. The consensus sequence identified for σE in vivo binding was similar to the one previously reported, except for a conserved G and A between the -35 and -10 regions. One third of the σE-binding sites did not contain the consensus sequence, suggesting there may be alternative mechanisms by which σE modulates transcription. By dissecting direct and indirect modes of σE-mediated regulation, we found that σE activates gene expression through recognition of both canonical and reversed consensus sequence. New σE regulated genes (greA, luxS, ompA and ompX) are shown to be involved in heat shock and oxidative stress responses.

Global analysis of Salmonella alternative sigma factor E on protein translation.

The alternative sigma factor E (σ(E)) is critical for response to extracytoplasmic stress in Salmonella. Extensive studies have been conducted on σ(E)-regulated gene expression, particularly at the transcriptional level. Increasing evidence suggests however that σ(E) may indirectly participate in post-transcriptional regulation. In this study, we conducted sample-matched global proteomic and transcriptomic analyses to determine the level of regulation mediated by σ(E) in Salmonella. Samples were analyzed from wild-type and isogenic rpoE mutant Salmonella cultivated in three different conditions: nutrient-rich and conditions that mimic early and late intracellular infection. We found that 30% of the observed proteome was regulated by σ(E) combining all three conditions. In different growth conditions, σ(E) affected the expression of a broad spectrum of Salmonella proteins required for miscellaneous functions. Those involved in transport and binding, protein synthesis, and stress response were particularly highlighted. By comparing transcriptomic and proteomic data, we identified genes post-transcriptionally regulated by σ(E) and found that post-transcriptional regulation was responsible for a majority of changes observed in the σ(E)-regulated proteome. Further, comparison of transcriptomic and proteomic data from hfq mutant of Salmonella demonstrated that σ(E)-mediated post-transcriptional regulation was partially dependent on the RNA-binding protein Hfq.

Analysis of the Salmonella regulatory network suggests involvement of SsrB and H-NS in σ(E)-regulated SPI-2 gene expression.

The extracytoplasmic functioning sigma factor σ(E) is known to play an essential role for Salmonella enterica serovar Typhimurium to survive and proliferate in macrophages and mice. However, its regulatory network is not well-characterized, especially during infection. Here we used microarray to identify genes regulated by σ(E) in Salmonella grown in three conditions: a nutrient-rich condition and two others that mimic early and late intracellular infection. We found that in each condition σ(E) regulated different sets of genes, and notably, several global regulators. When comparing nutrient-rich and infection-like conditions, large changes were observed in the expression of genes involved in Salmonella pathogenesis island (SPI)-1 type-three secretion system (TTSS), SPI-2 TTSS, protein synthesis, and stress responses. In total, the expression of 58% of Salmonella genes was affected by σ(E) in at least one of the three conditions. An important finding is that σ(E) up-regulates SPI-2 genes, which are essential for Salmonella intracellular survival, by up-regulating SPI-2 activator ssrB expression at the early stage of infection and down-regulating SPI-2 repressor hns expression at a later stage. Moreover, σ(E) is capable of countering the silencing of H-NS, releasing the expression of SPI-2 genes. This connection between σ(E) and SPI-2 genes, combined with the global regulatory effect of σ(E), may account for the lethality of rpoE-deficient Salmonella in murine infection.

A Novel Lactococcal Vaccine Expressing a Peptide from the M2 Antigen of H5N2 Highly Pathogenic Avian Influenza A Virus Prolongs Survival of Vaccinated Chickens.

A cost-effective and efficacious influenza vaccine for use in commercial poultry farms would help protect against avian influenza outbreaks. Current influenza vaccines for poultry are expensive and subtype specific, and therefore there is an urgent need to develop a universal avian influenza vaccine. We have constructed a live bacterial vaccine against avian influenza by expressing a conserved peptide from the ectodomain of M2 antigen (M2e) on the surface of Lactococcus lactis (LL). Chickens were vaccinated intranasally with the lactococcal vaccine (LL-M2e) or subcutaneously with keyhole-limpet-hemocyanin conjugated M2e (KLH-M2e). Vaccinated and nonvaccinated birds were challenged with high pathogenic avian influenza virus A subtype H5N2. Birds vaccinated with LL-M2e or KLH-M2e had median survival times of 5.5 and 6.0 days, respectively, which were significantly longer than non-vaccinated birds (3.5 days). Birds vaccinated subcutaneously with KLH-M2e had a lower mean viral burden than either of the other two groups. However, there was a significant correlation between the time of survival and M2e-specific serum IgG. The results of these trials show that birds in both vaccinated groups had significantly (P < 0.05) higher median survival times than non-vaccinated birds and that this protection could be due to M2e-specific serum IgG.

Bacterial resistance to antisense peptide phosphorodiamidate morpholino oligomers.

Peptide phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA mimics that bind cRNA and inhibit bacterial gene expression. The PPMO (RFF)(3)RXB-AcpP (where R is arginine, F, phenylalanine, X is 6-aminohexanoic acid, B is ß-alanine, and AcpP is acyl carrier protein) is complementary to 11 bases of the essential gene acpP (which encodes acyl carrier protein). The MIC of (RFF)(3)RXB-AcpP was 2.5 µM (14 µg/ml) in Escherichia coli W3110. The rate of spontaneous resistance of E. coli to (RFF)(3)RXB-AcpP was 4 × 10(-7) mutations/cell division. A spontaneous (RFF)(3)RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF)(3)RXB-AcpP was 40 µM (224 µg/ml) for PR200.1. The MICs of standard antibiotics for PR200.1 and W3110 were identical. The sequence of acpP was identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF)(3)RXB or peptides with a similar composition or pattern of cationic and nonpolar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF)(3)RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped to sbmA. Genetic complementation of PR200.1 or RR3 with sbmA restored susceptibility to (RFF)(3)RXB-AcpP. Deletion of sbmA caused resistance to (RFF)(3)RXB-AcpP. We conclude that resistance to (RFF)(3)RXB-AcpP was linked to the peptide and not the phosphorodiamidate morpholino oligomer, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF)(3)R-XB PPMOs may be transported across the plasma membrane by SbmA.