Utility of In Vivo Transcription Profiling for Identifying Pseudomonas aeruginosa Genes Needed for Gastrointestinal Colonization and Dissemination

Microarray analysis of Pseudomonas aeruginosa mRNA transcripts expressed in vivo during animal infection has not been previously used to investigate potential virulence factors needed in this setting. We compared mRNA expression in bacterial cells recovered from the gastrointestinal (GI) tracts of P. aeruginosa-colonized mice to that of P. aeruginosa in the drinking water used to colonize the mice. Genes associated with biofilm formation and type III secretion (T3SS) had markedly increased expression in the GI tract. A non-redundant transposon library in P. aeruginosa strain PA14 was used to test mutants in genes identified as having increased transcription during in vivo colonization. All of the Tn-library mutants in biofilm-associated genes had an attenuated ability to form biofilms in vitro, but there were no significant differences in GI colonization and dissemination between these mutants and WT P. aeruginosa PA14. To evaluate T3SS factors, we tested GI colonization and neutropenia-induced dissemination of both deletional (PAO1 and PAK) and insertional (PA14) mutants in four genes in the P. aeruginosa T3SS, exoS or exoU, exoT, and popB. There were no significant differences in GI colonization among these mutant strains and their WT counterparts, whereas rates of survival following dissemination were significantly decreased in mice infected by the T3SS mutant strains. However, there was a variable, strain-dependent effect on overall survival between parental and T3SS mutants. Thus, increased transcription of genes during in vivo murine GI colonization is not predictive of an essential role for the gene product in either colonization or overall survival following induction of neutropenia

Pore-Forming Toxins Induce Macrophage Necroptosis during Acute Bacterial Pneumonia

<div><p>Necroptosis is a highly pro-inflammatory mode of cell death regulated by RIP (or RIPK)1 and RIP3 kinases and mediated by the effector MLKL. We report that diverse bacterial pathogens that produce a pore-forming toxin (PFT) induce necroptosis of macrophages and this can be blocked for protection against <i>Serratia marcescens</i> hemorrhagic pneumonia. Following challenge with <i>S</i>. <i>marcescens</i>, <i>Staphylococcus aureus</i>, <i>Streptococcus pneumoniae</i>, <i>Listeria monocytogenes</i>, uropathogenic <i>Escherichia coli</i> (UPEC), and purified recombinant pneumolysin, macrophages pretreated with inhibitors of RIP1, RIP3, and MLKL were protected against death. Alveolar macrophages in MLKL KO mice were also protected during <i>S</i>. <i>marcescens</i> pneumonia. Inhibition of caspases had no impact on macrophage death and caspase-1 and -3/7 were determined to be inactive following challenge despite the detection of IL-1β in supernatants. Bone marrow-derived macrophages from RIP3 KO, but not caspase-1/11 KO or caspase-3 KO mice, were resistant to PFT-induced death. We explored the mechanisms for PFT-induced necroptosis and determined that loss of ion homeostasis at the plasma membrane, mitochondrial damage, ATP depletion, and the generation of reactive oxygen species were together responsible. Treatment of mice with necrostatin-5, an inhibitor of RIP1; GW806742X, an inhibitor of MLKL; and necrostatin-5 along with co-enzyme Q₁₀ (N5/C₁₀)_, which enhances ATP production; reduced the severity of <i>S</i>. <i>marcescens</i> pneumonia in a mouse intratracheal challenge model. N5/C₁₀ protected alveolar macrophages, reduced bacterial burden, and lessened hemorrhage in the lungs. We conclude that necroptosis is the major cell death pathway evoked by PFTs in macrophages and the necroptosis pathway can be targeted for disease intervention.</p></div

Inhibition of RIP1 or MLKL decreases morbidity and mortality during <i>S</i>. <i>marcescens</i> hemorrhagic pneumonia.

<p>BALB/c mice were infected intratracheally with <i>Sma</i> at high dose (5.0 x 10⁶ CFU) or low dose (1.0 x 10⁶ CFU). <b>A)</b> CFU recovered from the BALF of BALB/c mice pretreated with PBS or clodronate liposomes 24h after infection with low dose <i>Sma</i>. Each symbol represents an individual mouse. <b>B)</b> Survival of mice infected with the high dose of <i>Sma</i> that received intraperitoneal pre-treatment with CoQ_10, necrostatin-5 (N5), or N5 along with CoQ₁₀ (N5/C₁₀). Mice received 100 μl of a 100 μM solution of each drug intraperitoneally from time of challenge every 4h for the first 12h post-infection. <b>C)</b> Percent weight change and <b>D)</b> airway bacterial burden of <i>Sma</i> infected mice (n = 4–5/cohort) (low dose) when treated with PBS or the described N5/C₁₀ therapy. The concentration of <b>E)</b> total leukocytes, <b>F)</b> neutrophils, and <b>G)</b> monocytes (<i>y-</i>axis is log scale) in Hema-3-stained cytospins of BALF from <i>Sma</i> infected mice (low dose) that were received mock or N5/C₁₀ therapy. <b>H)</b> Airway bacterial burden, <b>I)</b> number of F4/80 positive cells in 50 μl, <b>J)</b> IL-1β levels, and <b>K)</b> TNFα levels in BALF from <i>Sma</i> infected mice (low dose) that received treatment with PBS, GW806742X (GW80; 100 μl of 100 μM), GSK’872 (100 μl of 10 μM) or ZVAD (100 μl of 10 μM). Mann-Whitney U tests were applied for two-group comparisons, for multiple group comparisons Dunn’s multiple-comparison post-test was used: *, <i>P</i> ≤ 0.05, **, <i>P</i> ≤ 0.01, ***, <i>P</i> ≤ 0.001. Data are representative of ≥3 separate experiments, each with 8 biological replicates.</p

RIP3 is required for PFT induced necroptosis.

<p><b>A)</b> LDH release assay of THP-1 cells transfected with siRNAs targeting RIP3, caspase-1 (Casp-1), caspase-8 (Casp-8) and a scramble control, infected with (A) <i>Sma</i> or challenged with <b>B)</b> recombinant pneumolysin (rPly). <b>C</b>) LDH release assay of MH-S macrophages infected with <i>Sma</i> at an MOI of 0.1 mock or following pretreatment with RIP3 inhibitor GSK’872. <b>D)</b> LDH release assay of BMDM from WT-C57BL/6, Caspase 1/11 KO, Caspase 3 KO and RIP3 KO mice, infected with <i>Sma</i> at an MOI of 0.5 or 0.1. <b>E)</b> LDH release of BMDM from wildtype mice, Caspase 1/11 (Casp1/11) KO, RIP3 KO, and BMDM from wildtype mice pretreated with necrostatin-5 (100μM) following their infection with <i>S</i>. <i>aureus</i> (MOI 10), <i>L</i>. <i>monocytogenes</i> (MOI 10) and <i>S</i>. <i>pneumoniae</i> (MOI 100). <b>F)</b> LDH release assay of BMDM from wild type C57BL/6 or RIP3 KO mice following their challenge with recombinant pneumolysin (rPly). <b>G)</b> LDH release assay of MH-S macrophages challenged with rPly with and without pretreatment with GSK’872 (10 μM). <b>H)</b> Alveolar macrophage numbers in BALF of RIP3 KO mice 18h after intratracheal infection with <i>S</i>. <i>marcescens</i>. Mann-Whitney tests were applied for two-group comparisons, for multiple group comparisons Dunn’s multiple-comparison post-test was used: *, <i>P</i> ≤ 0.05. Data are representative of ≥3 separate experiments, each with 8 biological replicates.</p

RIP1 and not caspases are required for <i>S</i>. <i>marcescens</i> induced macrophage cell death.

<p><b>A)</b> LDH release assay of MH-S macrophages infected with a high and low MOI of <i>S</i>. <i>marcescens</i> (<i>Sma</i>) following mock or pretreatment with the general caspase inhibitor Z-VAD-FMK (GI), caspase-1 inhibitor Z-WEHD-FMK (CI) caspase-3 inhibitor Z-DEVD-FMK (C3), caspase-8 inhibitor Z-YVAD-FMK (C8), and caspase-9 inhibitor Z-LEHD-FMK (C9), all at a 10 μM concentration. Percent positive cells for <b>B)</b> caspase-1 and <b>C)</b> caspase-3/7 activity 2h after <i>Sma</i> infection following FLICA staining and measured by FACs analyses. For controls, cells with nigericin induced pyroptosis (Ni +LPS; lipopolysaccharide at 10 ng/mL for 4h then Ni at 10 μM for 6 h) and cycloheximide (CHX; 2,000 μg/ml) induced apoptosis were measured, respectively. <b>D)</b> LDH release assay of MH-S macrophages infected with <i>Sma</i> at an MOI of 1 following their mock or pretreatment with necrostatin-1s, -1, -5, or -7 at a concentration of 100 μM. <b>E)</b> LDH release assay of MH-S macrophages infected with <i>Sma</i> after pretreatment with necrostatin-5 at increasing concentrations. <b>F)</b> LDH release assay of MH-S macrophages treated with CHX after pretreatment with necrostatin-5 (Nec 5; 100 μM) or ZVAD (10 μM). For multiple group comparisons Dunn’s multiple-comparison post-test was used: *, <i>P</i> ≤ 0.05, **, <i>P</i> ≤ 0.01, ***, <i>P</i> ≤ 0.001. Data are representative of ≥3 separate experiments, each with 8 biological replicates.</p

Mitochondrial damage and ATP depletion are required during PFT-induced necroptosis.

<p><b>A)</b> Cytochrome C (cyt C) levels in culture supernatants of MH-S cells infected with <i>S</i>. <i>marcescens</i> (<i>Sma</i>), <i>S</i>. <i>pneumoniae</i> (<i>Spn</i>), Δ<i>shlA</i>, and Δ<i>ply</i>. <b>B)</b> ATP levels in MH-S cell lysates following their infection with <i>Sma</i>, Δ<i>shlA</i>, and recombinant pneumolysin (rPly) or heat-inactivated rPly (HI-rPly) as measured using a luminescent cell viability assay. LDH release assay of MH-S cells infected with <i>Sma</i> following mock or pretreatment with <b>C)</b> soluble ATP (10 μg/mL), <b>D)</b> CoQ₁₀ (100μM), necrostatin-5 (N5; 100μM), N5/C₁₀ (100μM each), or <b>E)</b> resveratrol (Resv; 100μM). In vitro experiments were done at an MOI of 1 and 100 for <i>Sma</i> and <i>Spn</i>, respectively. Mann-Whitney U tests were applied for two-group comparisons, for multiple group comparisons Dunn’s multiple-comparison post-test was used: *, <i>P</i> ≤ 0.05, **, <i>P</i> ≤ 0.01, ***, <i>P</i> ≤ 0.001. Data are representative of ≥3 separate experiments; during LDH assay each with 8 biological replicates.</p