In vivo negative selection screen identifies genes required for Francisella virulence

Abstract

Francisella tularensis subverts the immune system to rapidly grow within mammalian hosts, often causing tularemia, a fatal disease. This pathogen targets the cytosol of macrophages where it replicates by using the genes encoded in the Francisella pathogenicity island. However, the bacteria are recognized in the cytosol by the host's ASC/caspase-1 pathway, which is essential for host defense, and leads to macrophage cell death and proinflammatory cytokine production. We used a microarray-based negative selection screen to identify Francisella genes that contribute to growth and/or survival in mice. The screen identified many known virulence factors including all of the Francisella pathogenicity island genes, LPS O-antigen synthetic genes, and capsule synthetic genes. We also identified 44 previously unidentified genes that were required for Francisella virulence in vivo, indicating that this pathogen may use uncharacterized mechanisms to cause disease. Among these, we discovered a class of Francisella virulence genes that are essential for growth and survival in vivo but do not play a role in intracellular replication within macrophages. Instead, these genes modulate the host ASC/caspase-1 pathway, a previously unidentified mechanism of Francisella pathogenesis. This finding indicates that the elucidation of the molecular mechanisms used by other uncharacterized genes identified in our screen will increase our understanding of the ways in which bacterial pathogens subvert the immune system. macrophage ͉ pathogenesis ͉ ASC ͉ caspase-1

Type I CRISPR-Cas targets endogenous genes and regulates virulence to evade mammalian host immunity

“…F. novicida Type II CRISPR-Cas system also regulates host inflammatory responses in host macrophages, but with notable differences that F. novicida Cas9 does not regulate bacteria's uptake by macrophages and F. novicida Cas9 impacts on immune response mediated by the IL-6/TLR2 signaling pathway [6,34]. During P. aeruginosa infection, early inflammatory signaling following NF-κB activation is dependent on both TLR2 and TLR4 [35], but here the inflammatory response permitted by the PA14 CRISPR-Cas-deletion mutant was primarily TLR4-dependent.…”

A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion

“…Murine bone marrow-derived macrophages were prepared from WT C57BL/6 mice or the indicated knockout strains and cultured as described previously (42). Macrophages were seeded overnight and infected with overnight cultures of the indicated bacterial strains at a MOI of 20:1 bacteria per macrophage.…”

“…F. novicida strain U112 and all derivatives used in this study were routinely grown at 37°C with aeration in tryptic soy broth (TSB) supplemented with 0.2% L-cysteine (BD Biosciences), or on tryptic soy agar plates supplemented with 0.1% L-cysteine. Cas9 regulatory axis deletion mutants and complementation strains were described previously (11,42). FTN_1254 and FTN_0109 mutants were constructed by allelic exchange as described previously (43,44) using primers in SI Appendix, Table S3.…”

A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1

“…The majority of these studies have used microarray technology to profile the transcriptional response of Francisella-infected immune cells (Andersson et al, 2006a, b;Butchar et al, 2008;Paranavitana et al, 2008). Some have used the microarraybased studies to identify virulence genes (Weiss et al, 2007) or study the control of virulence gene expression by regulators such as pmrA, mglA and sspA (Charity et The pdpA gene is one of the largest in the FPI and is located at the beginning of a putative operon containing the pdpB, vgrG and dotU genes. A previously described gene replacement mutant in the pdpA gene of F. novicida exhibited impaired intracellular replication and avirulence in mice; however, the substitution of the erythromycin resistance (Em R ) cassette for pdpA has since been shown by us (unpublished data) to have polarity effects on genes downstream of pdpA.…”