Brooke A. Napier

Subversion of Host Recognition and Defense Systems by Francisella spp.

SUMMARY 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.

Clinical Use of Colistin Induces Cross-Resistance to Host Antimicrobials in Acinetobacter baumannii

ABSTRACT The alarming rise in antibiotic resistance has led to an increase in patient mortality and health care costs. This problem is compounded by the absence of new antibiotics close to regulatory approval. Acinetobacter baumannii is a human pathogen that causes infections primarily in patients in intensive care units (ICUs) and is highly antibiotic resistant. Colistin is one of the last-line antibiotics for treating A. baumannii infections; however, colistin-resistant strains are becoming increasingly common. This cationic antibiotic attacks negatively charged bacterial membranes in a manner similar to that seen with cationic antimicrobials of the innate immune system. We therefore set out to determine if the increasing use of colistin, and emergence of colistin-resistant strains, is concomitant with the generation of cross-resistance to host cationic antimicrobials. We found that there is indeed a positive correlation between resistance to colistin and resistance to the host antimicrobials LL-37 and lysozyme among clinical isolates. Importantly, isolates obtained before and after treatment of individual patients demonstrated that colistin use correlated with increased resistance to cationic host antimicrobials. These data reveal the overlooked risk of inducing cross-resistance to host antimicrobials when treating patients with colistin as a last-line antibiotic. IMPORTANCE Increased use of the cationic antibiotic colistin to treat multidrug-resistant Acinetobacter baumannii has led to the development of colistin-resistant strains. Here we report that treatment of patients with colistin can induce not only increased resistance to colistin but also resistance to host cationic antimicrobials. This worrisome finding likely represents an example of a broader trend observed in other bacteria against which colistin is used therapeutically such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Furthermore, these data suggest that the possible future use of an array of cationic antimicrobial peptides in development as therapeutics may have unintended negative consequences, eventually leading to the generation of hypervirulent strains that are resistant to innate host defenses. The potential for the induction of cross-resistance to innate immune antimicrobials should be considered during the development of new therapeutics. Increased use of the cationic antibiotic colistin to treat multidrug-resistant Acinetobacter baumannii has led to the development of colistin-resistant strains. Here we report that treatment of patients with colistin can induce not only increased resistance to colistin but also resistance to host cationic antimicrobials. This worrisome finding likely represents an example of a broader trend observed in other bacteria against which colistin is used therapeutically such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Furthermore, these data suggest that the possible future use of an array of cationic antimicrobial peptides in development as therapeutics may have unintended negative consequences, eventually leading to the generation of hypervirulent strains that are resistant to innate host defenses. The potential for the induction of cross-resistance to innate immune antimicrobials should be considered during the development of new therapeutics.

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

Significance Increasing the integrity of the bacterial envelope is necessary to allow the successful survival of bacterial pathogens within the host and allow them to counteract damage caused by membrane-targeting antibiotics. We demonstrate that components of a clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) system, a prokaryotic defense against viruses and foreign nucleic acid, act to regulate the permeability of the bacterial envelope, ultimately providing these cells with the capability to resist membrane damage caused by antibiotics. This regulation further allows bacteria to resist detection by multiple host receptors to promote virulence. Overall, this study demonstrates the breadth of function of CRISPR-Cas systems in regulation, antibiotic resistance, innate immune evasion, and virulence. 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.

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 Francisellas largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is an important novel mediator of oxidative stress resistance.

Colistin heteroresistance in Enterobacter cloacae is associated with cross-resistance to the host antimicrobial lysozyme

ABSTRACT Here, we describe the first identification of colistin-heteroresistant Enterobacter cloacae in the United States. Treatment of this isolate with colistin increased the frequency of the resistant subpopulation and induced cross-resistance to the host antimicrobial lysozyme. This is the first description of heteroresistance conferring cross-resistance to a host antimicrobial and suggests that clinical treatment with colistin may inadvertently select for bacteria that are resistant to components of the host innate immune system.

Link between intraphagosomal biotin and rapid phagosomal escape in Francisella

Cytosolic bacterial pathogens require extensive metabolic adaptations within the host to replicate intracellularly and cause disease. In phagocytic cells such as macrophages, these pathogens must respond rapidly to nutrient limitation within the harsh environment of the phagosome. Many cytosolic pathogens escape the phagosome quickly (15–60 min) and thereby subvert this host defense, reaching the cytosol where they can replicate. Although a great deal of research has focused on strategies used by bacteria to resist antimicrobial phagosomal defenses and transiently pass through this compartment, the metabolic requirements of bacteria in the phagosome are largely uncharacterized. We previously identified a Francisella protein, FTN_0818, as being essential for intracellular replication and involved in virulence in vivo. We now show that FTN_0818 is involved in biotin biosynthesis and required for rapid escape from the Francisella-containing phagosome (FCP). Addition of biotin complemented the phagosomal escape defect of the FTN_0818 mutant, demonstrating that biotin is critical for promoting rapid escape during the short time that the bacteria are in the phagosome. Biotin also rescued the attenuation of the FTN_0818 mutant during infection in vitro and in vivo, highlighting the importance of this process. The key role of biotin in phagosomal escape implies biotin may be a limiting factor during infection. We demonstrate that a bacterial metabolite is required for phagosomal escape of an intracellular pathogen, providing insight into the link between bacterial metabolism and virulence, likely serving as a paradigm for other cytosolic pathogens.

Antibiotic failure mediated by a resistant subpopulation in Enterobacter cloacae.

Antibiotic resistance is a major public health threat, further complicated by unexplained treatment failures caused by bacteria that appear antibiotic susceptible. We describe an Enterobacter cloacae isolate harbouring a minor subpopulation that is highly resistant to the last-line antibiotic colistin. This subpopulation was distinct from persisters, became predominant in colistin, returned to baseline after colistin removal and was dependent on the histidine kinase PhoQ. During murine infection, but in the absence of colistin, innate immune defences led to an increased frequency of the resistant subpopulation, leading to inefficacy of subsequent colistin therapy. An isolate with a lower-frequency colistin-resistant subpopulation similarly caused treatment failure but was misclassified as susceptible by current diagnostics once cultured outside the host. These data demonstrate the ability of low-frequency bacterial subpopulations to contribute to clinically relevant antibiotic resistance, elucidating an enigmatic cause of antibiotic treatment failure and highlighting the critical need for more sensitive diagnostics.

A Francisella virulence factor catalyses an essential reaction of biotin synthesis

We recently identified a gene (FTN_0818) required for Francisella virulence that seemed likely involved in biotin metabolism. However, the molecular function of this virulence determinant was unclear. Here we show that this protein named BioJ is the enzyme of the biotin biosynthesis pathway that determines the chain length of the biotin valeryl side‐chain. Expression of bioJ allows growth of an Escherichia coli bioH strain on biotin‐free medium, indicating functional equivalence of BioJ to the paradigm pimeloyl‐ACP methyl ester carboxyl‐esterase, BioH. BioJ was purified to homogeneity, shown to be monomeric and capable of hydrolysis of its physiological substrate methyl pimeloyl‐ACP to pimeloyl‐ACP, the precursor required to begin formation of the fused heterocyclic rings of biotin. Phylogenetic analyses confirmed that distinct from BioH, BioJ represents a novel subclade of the α/β‐hydrolase family. Structure‐guided mapping combined with site‐directed mutagenesis revealed that the BioJ catalytic triad consists of Ser151, Asp248 and His278, all of which are essential for activity and virulence. The biotin synthesis pathway was reconstituted reaction in vitro and the physiological role of BioJ directly assayed. To the best of our knowledge, these data represent further evidence linking biotin synthesis to bacterial virulence.

Low Doses of Imatinib Induce Myelopoiesis and Enhance Host Anti-microbial Immunity

Imatinib mesylate (Gleevec) inhibits Abl1, c-Kit, and related protein tyrosine kinases (PTKs) and serves as a therapeutic for chronic myelogenous leukemia and gastrointestinal stromal tumors. Imatinib also has efficacy against various pathogens, including pathogenic mycobacteria, where it decreases bacterial load in mice, albeit at doses below those used for treating cancer. We report that imatinib at such low doses unexpectedly induces differentiation of hematopoietic stem cells and progenitors in the bone marrow, augments myelopoiesis but not lymphopoiesis, and increases numbers of myeloid cells in blood and spleen. Whereas progenitor differentiation relies on partial inhibition of c-Kit by imatinib, lineage commitment depends upon inhibition of other PTKs. Thus, imatinib mimics “emergency hematopoiesis,” a physiological innate immune response to infection. Increasing neutrophil numbers by adoptive transfer sufficed to reduce mycobacterial load, and imatinib reduced bacterial load of Franciscella spp., which do not utilize imatinib-sensitive PTKs for pathogenesis. Thus, potentiation of the immune response by imatinib at low doses may facilitate clearance of diverse microbial pathogens.

NaxD is a deacetylase required for lipid A modification and Francisella pathogenesis

Modification of specific Gram‐negative bacterial cell envelope components, such as capsule, O‐antigen and lipid A, are often essential for the successful establishment of infection. Francisella species express lipid A molecules with unique characteristics involved in circumventing host defences, which significantly contribute to their virulence. In this study, we show that NaxD, a member of the highly conserved YdjC superfamily, is a deacetylase required for an important modification of the outer membrane component lipid A in Francisella. Mass spectrometry analysis revealed that NaxD is essential for the modification of a lipid A phosphate with galactosamine in Francisella novicida, a model organism for the study of highly virulent Francisella tularensis. Significantly, enzymatic assays confirmed that this protein is necessary for deacetylation of its substrate. In addition, NaxD was involved in resistance to the antimicrobial peptide polymyxin B and critical for replication in macrophages and in vivo virulence. Importantly, this protein is also required for lipid A modification in F. tularensis as well as Bordetella bronchiseptica. Since NaxD homologues are conserved among many Gram‐negative pathogens, this work has broad implications for our understanding of host subversion mechanisms of other virulent bacteria.