DksA is a conserved master regulator of stress response in Acinetobacter baumannii.

Coordination of bacterial stress response mechanisms is critical for long-term survival in harsh environments for successful host infection. The general and specific stress responses of well-studied Gram-negative pathogens like Escherichia coli are controlled by alternative sigma factors, archetypically RpoS. The deadly hospital pathogen Acinetobacter baumannii is notoriously resistant to environmental stresses, yet it lacks RpoS, and the molecular mechanisms driving this incredible stress tolerance remain poorly defined. Here, using functional genomics, we identified the transcriptional regulator DksA as a master regulator for broad stress protection and virulence in A. baumannii. Transcriptomics, phenomics and in vivo animal studies revealed that DksA controls ribosomal protein expression, metabolism, mutation rates, desiccation, antibiotic resistance, and host colonization in a niche-specific manner. Phylogenetically, DksA was highly conserved and well-distributed across Gammaproteobacteria, with 96.6% containing DksA, spanning 88 families. This study lays the groundwork for understanding DksA as a major regulator of general stress response and virulence in this important pathogen.

Evolution of RND efflux pumps in the development of a successful pathogen.

AIMS: This study examined the origins and evolution of the AdeABC, AdeFGH and AdeIJK efflux pumps in the Acinetobacter genus, including human and animal pathogens and species from non-clinical environments. METHODS: Comparative genome analyses were performed using the reference sequences for 70 Acinetobacter species to identify putative orthologs of AdeABC, AdeFGH and AdeIJK and their regulators. Sequence similarities and the genomic locations of coding sequences were correlated with phylogeny to infer modes of evolution. Intraspecies variation was assessed in species of interest using up to 236 complete genome sequences. Mutants overproducing adeIJK in A. baylyi were examined to identify regulators of this system in a non A. baumannii species. RESULTS: The results indicate that adeIJK has been a stable part of Acinetobacter genomes since the genesis of this genus, whereas adeABC and adeFGH were carried by less than half of the species, but showed some lineage specificity. The organisation and local genetic contexts of adeABC loci were particularly variable to the sub-species level, suggesting frequent recombination. Cognate regulatory systems were almost always found in the genomes of species encoding pumps. Mutations in adeN, which encodes a repressor of adeIJK, were selected by antibiotic exposure in A. baylyi, similar to previous findings in pathogenic lineages. CONCLUSIONS: The multidrug efflux capacity of clinical Acinetobacter strains stems from accessory and core genetic features. AdeIJK is likely to have ancient core function(s) that have promoted its maintenance, whereas recent antibiotic use may be driving the evolution of the AdeABC pump.

Shigella flexneri remodeling and consumption of host lipids during infection.

IMPORTANCE: Bacterial pathogens have vastly distinct sites that they inhabit during infection. This requires adaptation due to changes in nutrient availability and antimicrobial stress. The bacterial surface is a primary barrier, and here, we show that the bacterial pathogen Shigella flexneri increases its surface decorations when it transitions to an intracellular lifestyle. We also observed changes in bacterial and host cell fatty acid homeostasis. Specifically, intracellular S. flexneri increased the expression of their fatty acid degradation pathway, while the host cell lipid pool was significantly depleted. Importantly, bacterial proliferation could be inhibited by fatty acid supplementation of host cells, thereby providing novel insights into the possible link between human malnutrition and susceptibility to S. flexneri.

Penicillin Binding Protein 7/8 Is a Potential Drug Target in Carbapenem-Resistant Acinetobacter baumannii.

Limited therapeutic options dictate the need for new classes of antimicrobials active against carbapenem-resistant Acinetobacter baumannii. Presented data confirm and extend penicillin binding protein 7/8 (PBP 7/8) as a high-value target in the CR A. baumannii strain HUMC1. PBP 7/8 was essential for optimal growth/survival of HUMC1 in ex vivo human ascites and in a rat subcutaneous abscess model; in a mouse pneumonia model, the absence of PBP 7/8 decreased lethality 11-fold. The loss of PBP 7/8 resulted in increased permeability, sensitivity to complement, and lysozyme-mediated bactericidal activity. These changes did not appear to be due to alterations in the cellular fatty acid composition or capsule production. However, a decrease in lipid A and an increase in coccoidal cells and cell aggregation were noted. The compromise of the stringent permeability barrier in the PBP 7/8 mutant was reflected by an increased susceptibility to several antimicrobials. Importantly, expression of ampC was not significantly affected by the loss of PBP 7/8 and serial passage of the mutant strain in human ascites over 7 days did not yield revertants possessing a wild-type phenotype. In summary, these data and other features support PBP 7/8 as a high-value drug target for extensively drug-resistant and CR A. baumannii. Our results guide next-stage studies; the determination that the inactivation of PBP 7/8 results in an increased sensitivity to lysozyme enables the design of a high-throughput screening assay to identify small molecule compounds that can specifically inhibit PBP 7/8 activity.

Physiological Functions of Bacterial "Multidrug" Efflux Pumps.

Bacterial multidrug efflux pumps have come to prominence in human and veterinary pathogenesis because they help bacteria protect themselves against the antimicrobials used to overcome their infections. However, it is increasingly realized that many, probably most, such pumps have physiological roles that are distinct from protection of bacteria against antimicrobials administered by humans. Here we undertake a broad survey of the proteins involved, allied to detailed examples of their evolution, energetics, structures, chemical recognition, and molecular mechanisms, together with the experimental strategies that enable rapid and economical progress in understanding their true physiological roles. Once these roles are established, the knowledge can be harnessed to design more effective drugs, improve existing microbial production of drugs for clinical practice and of feedstocks for commercial exploitation, and even develop more sustainable biological processes that avoid, for example, utilization of petroleum.

Identification of the Shigella flexneri Wzy Domain Modulating WzzpHS-2 Interaction and Detection of the Wzy/Wzz/Oag Complex.

Shigella flexneri implements the Wzy-dependent pathway to biosynthesize the O antigen (Oag) component of its surface lipopolysaccharide. The inner membrane polymerase WzySF catalyzes the repeat addition of undecaprenol-diphosphate-linked Oag (Und-PP-RUs) to produce a polysaccharide, the length of which is tightly regulated by two competing copolymerase proteins, WzzSF (short-type Oag; 10 to 17 RUs) and WzzpHS-2 (very-long-type Oag; >90 RUs). The nature of the interaction between WzySF and WzzSF/WzzpHS-2 in Oag polymerization remains poorly characterized, with the majority of the literature characterizing the individual protein constituents of the Wzy-dependent pathway. Here, we report instead a major investigation into the specific binding interactions of WzySF with its copolymerase counterparts. For the first time, a region of WzySF that forms a unique binding site for WzzpHS-2 has been identified. Specifically, this work has elucidated key WzySF moieties at the N- and C-terminal domains (NTD and CTD) that form an intramolecular pocket modulating the WzzpHS-2 interaction. Novel copurification data highlight that disruption of residues within this NTD-CTD pocket impairs the interaction with WzzpHS-2 without affecting WzzSF binding, thereby specifically disrupting polymerization of longer polysaccharide chains. This study provides a novel understanding of the molecular interaction of WzySF with WzzSF/WzzpHS-2 in the Wzy-dependent pathway and, furthermore, detects the Wzy/Wzz/Und-PP-Oag complex for the first time. Beyond S. flexneri, this work may be extended to provide insight into the interactions between protein homologues expressed by related species, especially members of Enterobacteriaceae, that produce dual Oag chain length determinants. IMPORTANCE Shigella flexneri is a pathogen causing significant morbidity and mortality, predominantly devastating the pediatric age group in developing countries. A major virulence factor contributing to S. flexneri pathogenesis is its surface lipopolysaccharide, which is comprised of three domains: lipid A, core oligosaccharide, and O antigen (Oag). The Wzy-dependent pathway is the most common biosynthetic mechanism implemented for Oag biosynthesis by Gram-negative bacteria, including S. flexneri. The nature of the interaction between the polymerase, WzySF, and the polysaccharide copolymerases, WzzSF and WzzpHS-2, in Oag polymerization is poorly characterized. This study investigates the molecular interplay between WzySF and its copolymerases, deciphering key interactions in the Wzy-dependent pathway that may be extended beyond S. flexneri, providing insight into Oag biosynthesis in Gram-negative bacteria.

Dietary zinc and the control of Streptococcus pneumoniae infection.

Human zinc deficiency increases susceptibility to bacterial infection. Although zinc supplementation therapies can reduce the impact of disease, the molecular basis for protection remains unclear. Streptococcus pneumoniae is a major cause of bacterial pneumonia, which is prevalent in regions of zinc deficiency. We report that dietary zinc levels dictate the outcome of S. pneumoniae infection in a murine model. Dietary zinc restriction impacts murine tissue zinc levels with distribution post-infection altered, and S. pneumoniae virulence and infection enhanced. Although the activation and infiltration of murine phagocytic cells was not affected by zinc restriction, their efficacy of bacterial control was compromised. S. pneumoniae was shown to be highly sensitive to zinc intoxication, with this process impaired in zinc restricted mice and isolated phagocytic cells. Collectively, these data show how dietary zinc deficiency increases sensitivity to S. pneumoniae infection while revealing a role for zinc as a component of host antimicrobial defences.

The Molecular Basis of Acinetobacter baumannii Cadmium Toxicity and Resistance.

Acinetobacter species are ubiquitous Gram-negative bacteria that can be found in water, in soil, and as commensals of the human skin. The successful inhabitation of Acinetobacter species in diverse environments is primarily attributable to the expression of an arsenal of stress resistance determinants, which includes an extensive repertoire of metal ion efflux systems. Metal ion homeostasis in the hospital pathogen Acinetobacter baumannii contributes to pathogenesis; however, insights into its metal ion transporters for environmental persistence are lacking. Here, we studied the impact of cadmium stress on A. baumannii. Our functional genomics and independent mutant analyses revealed a primary role for CzcE, a member of the cation diffusion facilitator (CDF) superfamily, in resisting cadmium stress. We also show that the CzcCBA heavy metal efflux system contributes to cadmium efflux. Collectively, these systems provide A. baumannii with a comprehensive cadmium translocation pathway from the cytoplasm to the periplasm and subsequently the extracellular space. Furthermore, analysis of the A. baumannii metallome under cadmium stress showed zinc depletion, as well as copper enrichment, both of which are likely to influence cellular fitness. Overall, this work provides new knowledge on the role of a broad arsenal of membrane transporters in A. baumannii metal ion homeostasis. IMPORTANCE Cadmium toxicity is a widespread problem, yet the interaction of this heavy metal with biological systems is poorly understood. Some microbes have evolved traits to proactively counteract cadmium toxicity, including Acinetobacter baumannii, which is notorious for persisting in harsh environments. Here, we show that A. baumannii utilizes a dedicated cadmium efflux protein in concert with a system that is primarily attuned to zinc efflux to efficiently overcome cadmium stress. The molecular characterization of A. baumannii under cadmium stress revealed how active cadmium efflux plays a key role in preventing the dysregulation of bacterial metal ion homeostasis, which appeared to be a primary means by which cadmium exerts toxicity upon the bacterium.

Structure and Metal Binding Properties of Chlamydia trachomatis YtgA.

The obligate intracellular pathogen Chlamydia trachomatis is a globally significant cause of sexually transmitted bacterial infections and the leading etiological agent of preventable blindness. The first-row transition metal iron (Fe) plays critical roles in chlamydial cell biology, and acquisition of this nutrient is essential for the survival and virulence of the pathogen. Nevertheless, how C. trachomatis acquires Fe from host cells is not well understood, since it lacks genes encoding known siderophore biosynthetic pathways, receptors for host Fe storage proteins, and the Fe acquisition machinery common to many bacteria. Recent studies have suggested that C. trachomatis directly acquires host Fe via the ATP-binding cassette permease YtgABCD. Here, we characterized YtgA, the periplasmic solute binding protein component of the transport pathway, which has been implicated in scavenging Fe(III) ions. The structure of Fe(III)-bound YtgA was determined at 2.0-Å resolution with the bound ion coordinated via a novel geometry (3 Ns, 2 Os [3N2O]). This unusual coordination suggested a highly plastic metal binding site in YtgA capable of interacting with other cations. Biochemical analyses showed that the metal binding site of YtgA was not restricted to interaction with only Fe(III) ions but could bind all transition metal ions examined. However, only Mn(II), Fe(II), and Ni(II) ions bound reversibly to YtgA, with Fe being the most abundant cellular transition metal in C. trachomatis Collectively, these findings show that YtgA is the metal-recruiting component of the YtgABCD permease and is most likely involved in the acquisition of Fe(II) and Mn(II) from host cells.IMPORTANCEChlamydia trachomatis is the most common bacterial sexually transmitted infection in developed countries, with an estimated global prevalence of 4.2% in the 15- to 49-year age group. Although infection is asymptomatic in more than 80% of infected women, about 10% of cases result in serious disease. Infection by C. trachomatis is dependent on the ability to acquire essential nutrients, such as the transition metal iron, from host cells. In this study, we show that iron is the most abundant transition metal in C. trachomatis and report the structural and biochemical properties of the iron-recruiting protein YtgA. Knowledge of the high-resolution structure of YtgA will provide a platform for future structure-based antimicrobial design approaches.

The Role of the CopA Copper Efflux System in Acinetobacter baumannii Virulence.

Acinetobacter baumannii has emerged as one of the leading causative agents of nosocomial infections. Due to its high level of intrinsic and adapted antibiotic resistance, treatment failure rates are high, which allows this opportunistic pathogen to thrive during infection in immune-compromised patients. A. baumannii can cause infections within a broad range of host niches, with pneumonia and bacteraemia being associated with the greatest levels of morbidity and mortality. Although its resistance to antibiotics is widely studied, our understanding of the mechanisms required for dealing with environmental stresses related to virulence and hospital persistence, such as copper toxicity, is limited. Here, we performed an in silico analysis of the A. baumannii copper resistome, examining its regulation under copper stress. Using comparative analyses of bacterial P-type ATPases, we propose that A. baumannii encodes a member of a novel subgroup of P1B-1 ATPases. Analyses of three putative inner membrane copper efflux systems identified the P1B-1 ATPase CopA as the primary mediator of cytoplasmic copper resistance in A. baumannii. Using a murine model of A. baumannii pneumonia, we reveal that CopA contributes to the virulence of A. baumannii. Collectively, this study advances our understanding of how A. baumannii deals with environmental copper toxicity, and it provides novel insights into how A. baumannii combats adversities encountered as part of the host immune defence.

The zinc efflux activator SczA protects Streptococcus pneumoniae serotype 2 D39 from intracellular zinc toxicity.

Zinc is an essential trace element that serves as a catalytic cofactor in metalloenzymes and a structural element in proteins involved in general metabolism and cellular defenses of pathogenic bacteria. Despite its importance, high zinc levels can impair cellular processes, inhibiting growth of many pathogenic bacteria, including the major respiratory pathogen Streptococcus pneumoniae. Zinc intoxication is prevented in S. pneumoniae by expression of the zinc exporter CzcD, whose expression is activated by the novel TetR-family transcriptional zinc-sensing regulator SczA. How zinc bioavailability triggers activation of SczA is unknown. It is shown here through functional studies in S. pneumoniae that an unannotated homodimeric TetR from S. agalactiae (PDB 3KKC) is the bona fide zinc efflux regulator SczA, and binds two zinc ions per protomer. Mutagenesis analysis reveals two metal binding sites, termed A and B, located on opposite sides of the SczA C-terminal regulatory domain. In vivo, the A- and B-site SczA mutant variants impact S. pneumoniae resistance to zinc toxicity and survival in infected macrophages. A model is proposed for S. pneumoniae SczA function in which both A- and B-sites were required for transcriptional activation of czcD expression, with the A-site serving as the evolutionarily conserved intracellular sensing site in SczAs.

Biotin-mediated growth and gene expression in Staphylococcus aureus is highly responsive to environmental biotin.

Biotin (Vitamin B7) is a critical enzyme co-factor in metabolic pathways important for bacterial survival. Biotin is obtained either from the environment or by de novo synthesis, with some bacteria capable of both. In certain species, the bifunctional protein BirA plays a key role in biotin homeostasis as it regulates expression of biotin biosynthetic enzymes in response to biotin demand and supply. Here, we compare the effect of biotin on the growth of two bacteria that possess a bifunctional BirA, namely Escherichia coli and Staphylococcus aureus. Unlike E. coli that could fulfill its biotin requirements through de novo synthesis, S. aureus showed improved growth rates in media supplemented with 10 nM biotin. S. aureus also accumulated more radiolabeled biotin from the media highlighting its ability to efficiently scavenge exogenous material. These data are consistent with S. aureus colonizing low biotin microhabitats. We also demonstrate that the S. aureus BirA protein is a transcriptional repressor of BioY, a subunit of the biotin transporter, and an operon containing yhfT and yhfS, the products of which have a putative role in fatty acid homeostasis. Increased expression of bioY is proposed to help cue S. aureus for efficient scavenging in low biotin environments.

Zinc stress induces copper depletion in Acinetobacter baumannii.

BACKGROUND: The first row transition metal ions zinc and copper are essential to the survival of many organisms, although in excess these ions are associated with significant toxicity. Here, we examined the impact of zinc and copper stress on Acinetobacter baumannii, a common opportunistic pathogen. RESULTS: We show that extracellular zinc stress induces a copper-specific depletion phenotype in A. baumannii ATCC 17978. Supplementation with copper not only fails to rescue this phenotype, but further exacerbates the copper depletion. Extensive analysis of the A. baumannii ATCC 17978 genome identified 13 putative zinc/copper resistance efflux pumps. Transcriptional analyses show that four of these transporters are responsive to zinc stress, five to copper stress and seven to the combination of zinc and copper stress, thereby revealing a likely foundation for the zinc-induced copper starvation in A. baumannii. In addition, we show that zinc and copper play crucial roles in management of oxidative stress and the membrane composition of A. baumannii. Further, we reveal that zinc and copper play distinct roles in macrophage-mediated killing of this pathogen. CONCLUSIONS: Collectively, this study supports the targeting of metal ion homeostatic mechanisms as an effective antimicrobial strategy against multi-drug resistant bacterial pathogens.

The First Histidine Triad Motif of PhtD Is Critical for Zinc Homeostasis in Streptococcus pneumoniae.

Streptococcus pneumoniae is the world's foremost human pathogen. Acquisition of the first row transition metal ion zinc is essential for pneumococcal colonization and disease. Zinc is acquired via the ATP-binding cassette transporter AdcCB and two zinc-binding proteins, AdcA and AdcAII. We have previously shown that AdcAII is reliant upon the polyhistidine triad (Pht) proteins to aid in zinc recruitment. Pht proteins generally contain five histidine (His) triad motifs that are believed to facilitate zinc binding and therefore play a significant role in pneumococcal metal ion homeostasis. However, the importance and potential redundancy of these motifs have not been addressed. We examined the effects of mutating each of the five His triad motifs of PhtD. The combination of in vitro growth assays, active zinc uptake, and PhtD expression studies show that the His triad closest to the protein's amino terminus is the most important for zinc acquisition. Intriguingly, in vivo competitive infection studies investigating the amino- and carboxyl-terminal His triad mutants indicate that the motifs have similar importance in colonization. Collectively, our new insights into the contributions of the individual His triad motifs of PhtD, and by extension the other Pht proteins, highlight the crucial role of the first His triad site in zinc acquisition. This study also suggests that the Pht proteins likely play a role beyond zinc acquisition in pneumococcal virulence.

Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins.

Chlorhexidine is widely used as an antiseptic or disinfectant in both hospital and community settings. A number of bacterial species display resistance to this membrane-active biocide. We examined the transcriptomic response of a representative nosocomial human pathogen, Acinetobacter baumannii, to chlorhexidine to identify the primary chlorhexidine resistance elements. The most highly up-regulated genes encoded components of a major multidrug efflux system, AdeAB. The next most highly overexpressed gene under chlorhexidine stress was annotated as encoding a hypothetical protein, named here as AceI. Orthologs of the aceI gene are conserved within the genomes of a broad range of proteobacterial species. Expression of aceI or its orthologs from several other γ- or ß-proteobacterial species in Escherichia coli resulted in significant increases in resistance to chlorhexidine. Additionally, disruption of the aceI ortholog in Acinetobacter baylyi rendered it more susceptible to chlorhexidine. The AceI protein was localized to the membrane after overexpression in E. coli. This protein was purified, and binding assays demonstrated direct and specific interactions between AceI and chlorhexidine. Transport assays using [(14)C]-chlorhexidine determined that AceI was able to mediate the energy-dependent efflux of chlorhexidine. An E15Q AceI mutant with a mutation in a conserved acidic residue, although unable to mediate chlorhexidine resistance and transport, was still able to bind chlorhexidine. Taken together, these data are consistent with AceI being an active chlorhexidine efflux protein and the founding member of a family of bacterial drug efflux transporters.

AdcA and AdcAII employ distinct zinc acquisition mechanisms and contribute additively to zinc homeostasis in Streptococcus pneumoniae.

Streptococcus pneumoniae is a globally significant human pathogen responsible for nearly 1 million deaths annually. Central to the ability of S. pneumoniae to colonize and mediate disease in humans is the acquisition of zinc from the host environment. Zinc uptake in S. pneumoniae occurs via the ATP-binding cassette transporter AdcCB, and, unusually, two zinc-binding proteins, AdcA and AdcAII. Studies have suggested that these two proteins are functionally redundant, although AdcA has remained uncharacterized by biochemical methods. Here we show that AdcA is a zinc-specific substrate-binding protein (SBP). By contrast with other zinc-binding SBPs, AdcA has two zinc-binding domains: a canonical amino-terminal cluster A-I zinc-binding domain and a carboxy-terminal zinc-binding domain, which has homology to the zinc-chaperone ZinT from Gram-negative organisms. Intriguingly, this latter feature is absent from AdcAII and suggests that the two zinc-binding SBPs of S. pneumoniae employ different modalities in zinc recruitment. We further show that AdcAII is reliant upon the polyhistidine triad proteins for zinc in vitro and in vivo. Collectively, our studies suggest that, despite the overlapping roles of the two SBPs in zinc acquisition, they may have unique mechanisms in zinc homeostasis and act in a complementary manner during host colonization.

Identification of genes essential for pellicle formation in Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii is an opportunistic pathogen, which has the ability to persist in the clinical environment, causing acute and chronic infections. A possible mechanism contributing to survival of A. baumannii is its ability to form a biofilm-like structure at the air/liquid interface, known as a pellicle. This study aimed to identify and characterise the molecular mechanisms required for pellicle formation in A. baumannii and to assess a broad range of clinical A. baumannii strains for their ability to form these multicellular structures. RESULTS: Random transposon mutagenesis was undertaken on a previously identified hyper-motile variant of A. baumannii ATCC 17978 designated 17978hm. In total three genes critical for pellicle formation were identified; cpdA, a phosphodiesterase required for degradation of cyclic adenosine monophosphate (cAMP), and A1S_0112 and A1S_0115 which are involved in the production of a secondary metabolite. While motility of the A1S_0112::Tn and A1S_0115::Tn mutant strains was abolished, the cpdA::Tn mutant strain displayed a minor alteration in its motility pattern. Determination of cAMP levels in the cpdA::Tn strain revealed a ~24-fold increase in cellular cAMP, confirming the role CpdA plays in catabolising this secondary messenger molecule. Interestingly, transcriptional analysis of the cpdA::Tn strain showed significant down-regulation of the operon harboring the A1S_0112 and A1S_0115 genes, revealing a link between these three genes and pellicle formation. Examination of our collection of 54 clinical A. baumannii strains revealed that eight formed a measurable pellicle; all of these strains were motile. CONCLUSIONS: This study shows that pellicle formation is a rare trait in A. baumannii and that a limited number of genes are essential for the expression of this phenotype. Additionally, an association between pellicle formation and motility was identified. The level of the signalling molecule cAMP was found to be controlled, in part, by the cpdA gene product, in addition to playing a critical role in pellicle formation, cellular hydrophobicity and motility. Furthermore, cAMP was identified as a novel regulator of the operon A1S_0112-0118.

Manganese uptake and streptococcal virulence.

Streptococcal solute-binding proteins (SBPs) associated with ATP-binding cassette transporters gained widespread attention first as ostensible adhesins, next as virulence determinants, and finally as metal ion transporters. In this mini-review, we will examine our current understanding of the cellular roles of these proteins, their contribution to metal ion homeostasis, and their crucial involvement in mediating streptococcal virulence. There are now more than 35 studies that have collected structural, biochemical and/or physiological data on the functions of SBPs across a broad range of bacteria. This offers a wealth of data to clarify the formerly puzzling and contentious findings regarding the metal specificity amongst this group of essential bacterial transporters. In particular we will focus on recent findings related to biological roles for manganese in streptococci. These advances will inform efforts aimed at exploiting the importance of manganese and manganese acquisition for the design of new approaches to combat serious streptococcal diseases.

Overlapping functionality of the Pht proteins in zinc homeostasis of Streptococcus pneumoniae.

Streptococcus pneumoniae is a globally significant pathogen that causes a range of diseases, including pneumonia, sepsis, meningitis, and otitis media. Its ability to cause disease depends upon the acquisition of nutrients from its environment, including transition metal ions such as zinc. The pneumococcus employs a number of surface proteins to achieve this, among which are four highly similar polyhistidine triad (Pht) proteins. It has previously been established that these proteins collectively aid in the delivery of zinc to the ABC transporter substrate-binding protein AdcAII. Here we have investigated the contribution of each individual Pht protein to pneumococcal zinc homeostasis by analyzing mutant strains expressing only one of the four pht genes. Under conditions of low zinc availability, each of these mutants showed superior growth and zinc accumulation profiles relative to a mutant strain lacking all four genes, indicating that any of the four Pht proteins are able to facilitate delivery of zinc to AdcAII. However, optimal growth and zinc accumulation in vitro and pneumococcal survival and proliferation in vivo required production of all four Pht proteins, indicating that, despite their overlapping functionality, the proteins are not dispensable without incurring a fitness cost. We also show that surface-attached forms of the Pht proteins are required for zinc recruitment and that they do not contribute to defense against extracellular zinc stress.

Comparative analysis of surface-exposed virulence factors of Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii is a significant hospital pathogen, particularly due to the dissemination of highly multidrug resistant isolates. Genome data have revealed that A. baumannii is highly genetically diverse, which correlates with major variations seen at the phenotypic level. Thus far, comparative genomic studies have been aimed at identifying resistance determinants in A. baumannii. In this study, we extend and expand on these analyses to gain greater insight into the virulence factors across eight A. baumannii strains which are clonally, temporally and geographically distinct, and includes an isolate considered non-pathogenic and a community-acquired A. baumannii. RESULTS: We have identified a large number of genes in the A. baumannii genomes that are known to play a role in virulence in other pathogens, such as the recently studied proline-alanine-alanine-arginine (PAAR)-repeat domains of the type VI secretion systems. Not surprising, many virulence candidates appear to be part of the A. baumannii core genome of virulent isolates but were often found to be insertionally disrupted in the avirulent A. baumannii strain SDF. Our study also reveals that many known or putative virulence determinants are restricted to specific clonal lineages, which suggests that these virulence determinants may be crucial for the success of these widespread common clones. It has previously been suggested that the high level of intrinsic and adaptive resistance has enabled the widespread presence of A. baumannii in the hospital environment. This appears to have facilitated the expansion of its repertoire of virulence traits, as in general, the nosocomial strains in this study possess more virulence genes compared to the community-acquired isolate. CONCLUSIONS: Major genetic variation in known or putative virulence factors was seen across the eight strains included in this study, suggesting that virulence mechanisms are complex and multifaceted in A. baumannii. Overall, these analyses increase our understanding of A. baumannii pathogenicity and will assist in future studies determining the significance of virulence factors within clonal lineages and/or across the species.