SummaryThe Dot/Icm type IV secretion system of Legionella pneumophila triggers robust activation of caspase-3 during early and exponential stages of proliferation within human macrophages, but apoptosis is delayed till late stages of infection, which is novel. As caspase-3 is the executioner of the cell, we tested the hypothesis that L. pneumophila triggers antiapoptotic signalling within the infected human macrophages to halt caspase-3 from dismantling the cells. Here we show that during early and exponential replication, L. pneumophila-infected human monocyte-derived macrophages (hMDMs) exhibit a remarkable resistance to induction of apoptosis, in a Dot/Icm-dependent manner. Microarray analyses and real-time PCR reveal that during exponential intracellular replication, L. pneumophila triggers upregulation of 12 anti-apoptotic genes that are linked to activation of the nuclear transcription factor kappa-B (NF-kB). Our data show that L. pneumophila induces a Dot/Icm-dependent sustained nuclear translocation of the p50 and p65 subunits of NF-kB during exponential intracellular replication. Bacterial entry is essential both for the anti-apoptotic phenotype of infected hMDMs and for nuclear translocation of the p65. Using p65 -/-and IKKa -/-b -/-double knockout mouse embryonic fibroblast cell lines, we show that nuclear translocation of NF-kB is required for the resistance of L. pneumophila-infected cells to apoptosis-inducing agents. In addition, the L. pneumophila-induced nuclear translocation of NF-kB requires the activity of IKKa and/or IKKb. We conclude that although the Dot/Icm secretion system of L. pneumophila elicits an early robust activation of caspase-3 in human macrophages, it triggers a strong anti-apoptotic signalling cascade mediated, at least in part by NF-kB, which renders the cells refractory to external potent apoptotic stimuli.

SummaryThe Dot/Icm type IV secretion system of Legionella pneumophila is essential for evasion of endocytic fusion and for activation of caspase-3 during early stages of infection of macrophages, but the mechanisms of manipulating these host cell processes are not known. Here, we show that caspase-3 activation by L. pneumophila is independent of all the known apoptotic pathways that converge on the activation of caspase-3. The cytoplasmic proteins IcmS, IcmR and IcmQ, which are involved in secretion of Dot/Icm effectors, are required for caspase-3 activation. Pretreatment of U937 macrophages and human peripheral blood monocytes (hPBM) with the capase-3 inhibitor (DEVD-fmk) or the paninhibitor of caspases (Z-VAD-fmk) before infection blocks intracellular replication of L. pneumophila in a dose-dependent manner. Inhibition of caspase-3 results in co-localization of the L. pneumophila -containing phagosome (LCP) with the late endosomal/lysosomal marker Lamp-2, and the LCP contains lysosomal enzymes, similar to the dotA mutant, which is defective in caspase-3 activation. However, activation of caspase-3 before infection does not rescue the replication defect of the dotA mutant. Interestingly, inhibition of caspase-3 after a 15 or 30 min infection period by the parental strain has no detectable effect on the formation of a replicative niche. The Dot/Icm-mediated activation of caspase-3 by L. pneumophila specifically cleaves, in a dose-and time-dependent manner, the Rab5 effector Rabaptin-5, which maintains Rab5-GTP on the endosomal membrane. In addition, PI3 kinase, which is a crucial effector of Rab5 downstream of Rababptin-5, is not required for intracellular replication. Using single-cell analysis, we show that apoptosis is not evident in the infected cell until bacterial replication results in > 20 bacteria per cell. We conclude that activation of caspase-3 by the Dot/Icm virulence system of L. pneumophila is essential for halting biogenesis of the LCP through the endosomal/lysosomal pathway, and that this is associated with the cleavage of Rabpatin-5.

The ability of the intracellular bacterium Legionella pneumophila to cause disease is totally dependent on its ability to modulate the biogenesis of its phagosome and to replicate within alveolar cells. Upon invasion, L. pneumophila activates caspase-3 in macrophages, monocytes, and alveolar epithelial cells in a Dot/Icmdependent manner that is independent of the extrinsic or intrinsic pathway of apoptosis, suggesting a novel mechanism of caspase-3 activation by this intracellular pathogen. We have shown that the inhibition of caspase-3 prior to infection results in altered biogenesis of the L. pneumophila-containing phagosome and in an inhibition of intracellular replication. In this report, we show that the preactivation of caspase-3 prior to infection does not rescue the intracellular replication of L. pneumophila icmS, icmR, and icmQ mutant strains. Interestingly, preactivation of caspase-3 through the intrinsic and extrinsic pathways of apoptosis in both human and mouse macrophages inhibits intracellular replication of the parental stain of L. pneumophila. Using single-cell analysis, we show that intracellular L. pneumophila induces a robust activation of caspase-3 during exponential replication. Surprisingly, despite this robust activation of caspase-3 in the infected cell, the host cell does not undergo apoptosis until late stages of infection. In sharp contrast, the activation of caspase-3 by apoptosis-inducing agents occurs concomitantly with the apoptotic death of all cells that exhibit caspase-3 activation. It is only at a later stage of infection, and concomitant with the termination of intracellular replication, that the L. pneumophila-infected cells undergo apoptotic death. We conclude that although a robust activation of caspase-3 is exhibited throughout the exponential intracellular replication of L. pneumophila, apoptotic cell death is not executed until late stages of the infection, concomitant with the termination of intracellular replication.

The induction of virulence traits by Legionella pneumophila at the post-exponential phase has been proposed to be triggered by the stringent response mediated by RelA, which triggers RpoS. We show that L. pneumophila rpoS but not relA is required for early intracellular survival and replication within human monocyte-derived macrophages and Acanthamoeba polyphaga. In addition, L. pneumophila rpoS but not relA is required for expression of the pore-forming activity. We provide evidence that RpoS plays a role in the modulation of phagosome biogenesis and in adaptation to the phagosomal microenvironment. Thus, there is no functional link between the stringent response and RpoS in the pathogenesis of L. pneumophila.

This report presents the prevalence of Palestinian isolates of methicillin-resistant Staphylococcus aureus (MRSA) in nosocomial infections and their antibiotic resistant pattern. A total of 321 clinical isolates of S. aureus were identified from different patients. The prevalence of methicillin resistance among S. aureus isolates was 8.7% (28 isolates). Resistance rates of MRSA to other antibiotics were as follows: 82.1% resistant to erythromycin, 67.9% to clindamycin, 64.3% to gentamicin, and 32.1% to ciprofloxacin. No co-trimoxazole- and vancomycin-resistant isolates were identified in this study. The proportion of methicillin resistance was highest among S. aureus isolates associated with upper respiratory specimens (42.8%); the proportion of methicillin resistance was 39.3% among skin ulcer isolates, 10.7% among urinary tract infection isolates, and lowest among isolates associated with blood and prostate discharge (3.6% each).

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Background Antimicrobial resistance is a worldwide threat to public health. WHO has created several resolutions and strategies on this subject at the World Health Assembly. In May, 2015, WHO published a global action plan to mitigate antimicrobial resistance, including tracking and global surveillance focusing on improving awareness and understanding of this issue. The aim of this study was to screen for carbapenem-resistant bacteria in the occupied Palestinian Territory, to investigate the mechanisms behind the resistance, and to assess the scope of this difficulty in the area.Methods During 6 weeks in 2012, we collected all available Gram-negative isolates taken from inpatients and outpatients in hospital laboratories at Al-Shifa Hospital and five additional hospitals in the West Bank to screen for carbapenem resistance. Resistant isolates were identified with MALDI-TOF, mapped for their resistance pattern, and further analysed for mechanism of resistance by multiplex PCR and gene sequencing. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to type bacteria to compare the resistant isolates locally and internationally.Findings Of the 248 Gram-negative isolates we collected, 21 (8%) showed significant in-vitro resistance to carbapenems and several other antibiotics. These 21 were identified as 15 isolates of Acinetobacter baumannii and six of Pseudomonas aeruginosa. Carbapenemase gene investigations showed intrinsic OXA-51 group in all isolates and one isolate from Gaza was positive for NDM-2. Of the six P aeruginosa isolates, one VIM-4 and three VIM-2 producers were recorded and MLST reported three new sequence types named ST1562, ST1563, and ST1564.Interpretation We identified a high ratio of multidrug-resistant bacteria in the occupied Palestinian territory, to our knowledge the first documented isolates showing production of NDM-2 and VIM carbapenemases as a contributing mechanism. These findings emphasise the importance of this growing health threat in the occupied Palestinian territory and the need for further investigation and adequate surveillance of antibiotic resistance.

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