In Vivo Fitness Adaptations of Colistin-Resistant Acinetobacter baumannii Isolates to Oxidative Stress.

The loss of fitness in colistin-resistant (CR) Acinetobacter baumannii was investigated using longitudinal isolates from the same patient. Early CR isolates were outcompeted by late CR isolates for growth in broth and survival in the lungs of mice. Fitness loss was associated with an increased susceptibility to oxidative stress since early CR strains had reduced in vitro survival in the presence of hydrogen peroxide and decreased catalase activity compared to that of late CR and colistin-susceptible (CS) strains.

Fatal outbreak of an emerging clone of extensively drug-resistant Acinetobacter baumannii with enhanced virulence.

BACKGROUND: Severe Acinetobacter baumannii infections in immunocompetent patients are uncommon, and the virulence mechanisms of this organism are not fully understood. METHODS: Following an outbreak of fatal A. baumannii infections in a cohort of relatively immunocompetent patients (low comorbidity and illness severity scores), isolates were investigated with comparative genomics and in animal models. RESULTS: Two unrelated A. baumannii clades were associated with the outbreak. The clone associated with the majority of patient deaths, clade B, is evolutionarily distinct from the 3 international clonal complexes, belongs to multilocus sequence type (MLST) 10, and is most closely related to strains isolated from the Czech Republic, California, and Germany in 1994, 1997, and 2003, respectively. In 2 different murine models, clade B isolates were more virulent than comparator strains, including the highly virulent reference strain AB5075. The most virulent clade B derivative, MRSN 16897, was isolated from the patient with the lowest combined comorbidity/illness severity score. Clade B isolates possess a unique combination of putative virulence genes involved in iron metabolism, protein secretion, and glycosylation, which was leveraged to develop a rapid and specific clinical assay to detect this clade that cannot be distinguished by MLST. CONCLUSIONS: Clade B warrants continued surveillance and investigation.

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

Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.

Staphylococcus aureus hyaluronidase is a CodY-regulated virulence factor.

Staphylococcus aureus is a Gram-positive pathogen that causes a diverse range of bacterial infections. Invasive S. aureus strains secrete an extensive arsenal of hemolysins, immunomodulators, and exoenzymes to cause disease. Our studies have focused on the secreted enzyme hyaluronidase (HysA), which cleaves the hyaluronic acid polymer at the ß-1,4 glycosidic bond. In the study described in this report, we have investigated the regulation and contribution of this enzyme to S. aureus pathogenesis. Using the Nebraska Transposon Mutant Library (NTML), we identified eight insertions that modulate extracellular levels of HysA activity. Insertions in the sigB operon, as well as in genes encoding the global regulators SarA and CodY, significantly increased HysA protein levels and activity. By altering the availability of branched-chain amino acids, we further demonstrated CodY-dependent repression of HysA activity. Additionally, through mutation of the CodY binding box upstream of hysA, the repression of HysA production was lost, suggesting that CodY is a direct repressor of hysA expression. To determine whether HysA is a virulence factor, a ΔhysA mutant of a community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 strain was constructed and found to be attenuated in a neutropenic, murine model of pulmonary infection. Mice infected with this mutant strain exhibited a 4-log-unit reduction in bacterial burden in their lungs, as well as reduced lung pathology and increased levels of pulmonary hyaluronic acid, compared to mice infected with the wild-type, parent strain. Taken together, these results indicate that S. aureus hyaluronidase is a CodY-regulated virulence factor.

Induction of human plasmablasts during infection with antibiotic-resistant nosocomial bacteria.

OBJECTIVES: Nosocomial pathogens such as Acinetobacter baumannii are a growing public health threat, due in part to their increasing resistance to antibiotics. Since some strains are resistant to all available antibiotics, novel therapies are urgently needed. Plasmablasts are short-lived B cells found in the blood that can be collected and harnessed to produce therapeutic antibodies. We set out to determine whether plasmablasts are induced during infection with A. baumannii and other nosocomial pathogens. METHODS: We obtained blood samples from patients infected with antibiotic-resistant nosocomial pathogens, and analysed their plasmablast response by flow cytometry. RESULTS: We observed a strong induction of plasmablasts in patients with antibiotic-resistant A. baumannii infection. Furthermore, plasmablasts were also induced in response to other drug-resistant nosocomial pathogens. CONCLUSIONS: These data suggest that plasmablasts may be broadly harnessed to develop therapeutic antibodies to combat otherwise untreatable antibiotic-resistant infections.

Subversion of host recognition and defense systems by Francisella spp.

Francisella tularensis is a gram-negative intracellular pathogen and the causative agent of the disease tularemia. Inhalation of as few as 10 bacteria is sufficient to cause severe disease, making F. tularensis one of the most highly virulent bacterial pathogens. The initial stage of infection is characterized by the "silent" replication of bacteria in the absence of a significant inflammatory response. Francisella achieves this difficult task using several strategies: (i) strong integrity of the bacterial surface to resist host killing mechanisms and the release of inflammatory bacterial components (pathogen-associated molecular patterns [PAMPs]), (ii) modification of PAMPs to prevent activation of inflammatory pathways, and (iii) active modulation of the host response by escaping the phagosome and directly suppressing inflammatory pathways. We review the specific mechanisms by which Francisella achieves these goals to subvert host defenses and promote pathogenesis, highlighting as-yet-unanswered questions and important areas for future study.

Repression of bacterial lipoprotein production by Francisella novicida facilitates evasion of innate immune recognition.

Innate recognition systems, including the Toll-like receptors (TLRs), play a critical role in activating host defences and proinflammatory pathways in response to infection. Pathogens have developed strategies to subvert TLRs in order to survive and replicate within the host. The model intracellular pathogen, Francisella novicida, modulates host defences to promote survival and replication in macrophages. TLR2, which recognizes bacterial lipoproteins (BLPs), is critical for activating host defences and proinflammatory cytokine production in response to Francisella infection. Here we show that the F. novicida protein FTN_0757 acts to repress BLP production, dampening TLR2 activation. The ΔFTN_0757 mutant strain induced robust TLR2-dependent cytokine production in macrophages compared with wild-type bacteria, and produced increased amounts of BLPs. The deletion of one BLP (FTN_1103) from ΔFTN_0757 decreased the total BLP concentration to near wild-type level sand correlated with a decrease in the inductionof TLR2 signalling. The overproduction of BLPs also contributed to the in vivo attenuation of the ΔFTN_0757 mutant, which was significantly rescued when FTN_1103 was deleted. Taken together, these data reveal a novel mechanism of immune evasion by the downregulation of BLP expression to subvert TLR2 activation, which is likely used by numerous other intracellular bacterial pathogens.

Macrophage replication screen identifies a novel Francisella hydroperoxide resistance protein involved in virulence.

Francisella tularensis is a gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI), validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is an important novel mediator of oxidative stress resistance.

TLR2 signaling contributes to rapid inflammasome activation during F. novicida infection.

BACKGROUND: Early detection of microorganisms by the innate immune system is provided by surface-expressed and endosomal pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). Detection of microbial components by TLRs initiates a signaling cascade leading to the expression of proinflammatory cytokines including IL-6 and IL-1ß. Some intracellular bacteria subvert the TLR response by rapidly escaping the phagosome and entering the cytosol. However, these bacteria may be recognized by the inflammasome, a multi-protein complex comprised of a sensor protein, ASC and the cysteine protease caspase-1. Inflammasome activation leads to release of the proinflammatory cytokines IL-1ß and IL-18 and death of the infected cell, an important host defense that eliminates the pathogen's replicative niche. While TLRs and inflammasomes are critical for controlling bacterial infections, it is unknown whether these distinct host pathways cooperate to activate defenses against intracellular bacteria. METHODOLOGY/SIGNIFICANT FINDINGS: Using the intracellular bacterium Francisella novicida as a model, we show that TLR2(-/-) macrophages exhibited delayed inflammasome activation compared to wild-type macrophages as measured by inflammasome assembly, caspase-1 activation, cell death and IL-18 release. TLR2 also contributed to inflammasome activation in response to infection by the cytosolic bacterium Listeria monocytogenes. Components of the TLR2 signaling pathway, MyD88 and NF-κB, were required for rapid inflammasome activation. Furthermore, TLR2(-/-) mice exhibited lower levels of cell death, caspase-1 activation, and IL-18 production than wild-type mice upon F. novicida infection. CONCLUSIONS/SIGNIFICANCE: These results show that TLR2 is required for rapid inflammasome activation in response to infection by cytosolic bacterial pathogens. In addition to further characterizing the role of TLR2 in host defense, these findings broaden our understanding of how the host integrates signals from spatiotemporally separated PRRs to coordinate an innate response against intracellular bacteria.