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.

Characterizing the role of phosphatidylglycerol-phosphate phosphatases in Acinetobacter baumannii cell envelope biogenesis and antibiotic resistance.

The dissemination of multi-drug resistant Acinetobacter baumannii threatens global healthcare systems and necessitates the development of novel therapeutic options. The Gram-negative bacterial cell envelope provides a first defensive barrier against antimicrobial assault. Essential components of this multi-layered complex are the phospholipid-rich membranes. Phosphatidylglycerol phosphate (PGP) phosphatases are responsible for a key step in the biosynthesis of a major phospholipid species, phosphatidylglycerol (PG), but these enzymes have also been implicated in the biogenesis of other cell envelope components. Our bioinformatics analyses identified two putative PGP candidates in the A. baumannii genome, PgpA and PgpB. Phospholipid analyses of isogenic pgpA mutants in two distinct A. baumannii strains revealed a shift in the desaturation levels of phosphatidylethanolamine (PE) phospholipid species, possibly due to the activation of the phospholipid desaturase DesA. We also investigated the impact of the inner membrane phosphatases on other cell envelope components, which revealed a role of PgpB in the maintenance of the A. baumannii peptidoglycan layer, and consequently carbapenem resistance. Collectively, this work provides novel insights into the roles of PGP phosphatases on the global lipidomic landscape of A. baumannii and their interconnectivity with the biogenesis of other cell envelope components. The non-essentiality of these candidates exemplifies metabolic versatility of A. baumannii, which is believed to be key to its success as global pathogen.

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.

The Impact of Omega-3 Fatty Acids on the Evolution of Acinetobacter baumannii Drug Resistance.

The bacterial pathogen Acinetobacter baumannii has emerged as an urgent threat to health care systems. The prevalence of multidrug resistance in this critical human pathogen is closely associated with difficulties in its eradication from the hospital environment and its recalcitrance to treatment during infection. The development of resistance in A. baumannii is in part due to substantial plasticity of its genome, facilitating spontaneous genomic evolution. Many studies have investigated selective pressures imposed by antibiotics on genomic evolution, but the influence of high-abundance bioactive molecules at the host-pathogen interface on mutation and rates of evolution is poorly understood. Here, we studied the roles of host fatty acids in the gain in resistance to common antibiotics. We defined the impact of the polyunsaturated fatty acids arachidonic acid and docosahexaenoic acid on the development of resistance to erythromycin in A. baumannii strain AB5075_UW using a microevolutionary approach. We employed whole-genome sequencing and various phenotypic analyses to characterize microbe-lipid-antibiotic interactions. Cells exposed to erythromycin in the presence of the fatty acids displayed significantly lower rates of development of resistance to erythromycin and, importantly, tetracycline. Subsequent analyses defined diverse means by which host fatty acids influence the mutation rates. This work has highlighted the critical need to consider the roles of host fatty acids in A. baumannii physiology and antimicrobial resistance. Collectively, we have identified a novel means to curb the development of resistance in this critical human pathogen. IMPORTANCE The global distribution of multidrug resistance in A. baumannii has necessitated seeking not only alternative therapeutic approaches but also the means to limit the development of resistance in clinical settings. Highly abundant host bioactive compounds, such as polyunsaturated fatty acids, are readily acquired by A. baumannii during infection and have been illustrated to impact the bacterium's membrane composition and antibiotic resistance. In this work, we show that in vitro supplementation with host polyunsaturated fatty acids reduces the rate at which A. baumannii gains resistance to erythromycin and tetracycline. Furthermore, we discover that the impact on resistance development is closely associated with the primary antimicrobial efflux systems of A. baumannii, which represent one of the major drivers of clinical resistance. Overall, this study emphasizes the potential of host macromolecules in novel approaches to circumvent the difficulties of multidrug resistance during A. baumannii treatment, with fatty acid supplements such as fish oil providing safe and cost-effective ways to enhance host tolerance to bacterial infections.

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.

The Acinetobacter baumannii Autotransporter Adhesin Ata Recognizes Host Glycans as High-Affinity Receptors.

Acinetobacter baumannii is a significant opportunistic pathogen responsible for infections of the lung, blood, skin, urinary tract, and soft tissues, with some strains exhibiting almost complete resistance to commonly used antibiotics. This multidrug resistance, together with a dearth of new antibiotic development, mean novel methods of treatment and prevention are urgently needed. Although many A. baumannii factors required to colonize the host have been identified, little is known about the specific host molecules recognized by these factors. A. baumannii produces a trimeric autotransporter adhesin known as Ata that has been previously demonstrated to bind components of the host cell's extracellular matrix, which are often heavily glycosylated. We hypothesized that Ata would exhibit lectin activity which would play a role in adherence to the host cell surface. Our biophysical analysis using glycan arrays and surface plasmon resonance demonstrated that Ata binds galactose, N-acetylglucosamine, and galactose (ß1-3/4) N-acetylglucosamine with high-affinity. These structures are present on many of the proteins which were previously reported to be bound by Ata. We also demonstrated that the recognition of human plasma fibronectin by Ata requires this ability to bind glycans, as the interaction between Ata and fibronectin does not occur when fibronectin is deglycosylated. This strongly suggests a key role for Ata lectin activity during host adherence. This information will assist in directing the development of new and effective treatments to block host interactions using glycans and/or novel compounds in multidrug resistant A. baumannii infections.

The Membrane Composition Defines the Spatial Organization and Function of a Major Acinetobacter baumannii Drug Efflux System.

Acinetobacter baumannii is one of the world's most problematic nosocomial pathogens. The combination of its intrinsic resistance and ability to acquire resistance markers allow this organism to adjust to antibiotic treatment. Despite being the primary barrier against antibiotic stress, our understanding of the A. baumannii membrane composition and its impact on resistance remains limited. In this study, we explored how the incorporation of host-derived polyunsaturated fatty acids (PUFAs) is associated with increased antibiotic susceptibility. Functional analyses of primary A. baumannii efflux systems indicated that AdeB-mediated antibiotic resistance was impacted by PUFA treatment. Molecular dynamics simulations of AdeB identified a specific morphological disruption of AdeB when positioned in the PUFA-enriched membrane. Collectively, we have shown that PUFAs can impact antibiotic efficacy via a vital relationship with antibiotic efflux pumps. Furthermore, this work has revealed that A. baumannii's unconditional desire for fatty acids may present a possible weakness in its multidrug resistance capacity. IMPORTANCE Antimicrobial resistance is an emerging global health crisis. Consequently, we have a critical need to prolong our current arsenal of antibiotics, in addition to the development of novel treatment options. Due to their relatively high abundance at the host-pathogen interface, PUFAs and other fatty acid species not commonly synthesized by A. baumannii may be actively acquired by A. baumannii during infection and change the biophysical properties of the membrane beyond that studied in standard laboratory culturing media. Our work illustrates how the membrane phospholipid composition impacts membrane protein function, which includes an important multidrug efflux system in extensively-drug-resistant A. baumannii. This work emphasizes the need to consider including host-derived fatty acids in in vitro analyses of A. baumannii. On a broader scope, this study presents new findings on the potential health benefits of PUFA in individuals at risk of contracting A. baumannii infections or those undergoing antibiotic treatment.

To Make or Take: Bacterial Lipid Homeostasis during Infection.

Bacterial fatty acids are critical components of the cellular membrane. A shift in environmental conditions or in the bacterium's lifestyle may result in the requirement for a distinct pool of fatty acids with unique biophysical properties. This can be achieved by the modification of existing fatty acids or via de novo synthesis. Furthermore, bacteria have evolved efficient means to acquire these energy-rich molecules from their environment. However, the balance between de novo fatty acid synthesis and exogenous acquisition during pathogenesis is poorly understood. Here, we studied the mouse fatty acid landscape prior to and after infection with Acinetobacter baumannii, a Gram-negative, opportunistic human pathogen. The lipid fluxes observed following infection revealed fatty acid- and niche-specific changes. Lipidomic profiling of A. baumannii isolated from the pleural cavity of mice identified novel A. baumannii membrane phospholipid species and an overall increased abundance of unsaturated fatty acid species. Importantly, we found that A. baumannii relies largely upon fatty acid acquisition in all but one of the studied niches, the blood, where the pathogen biosynthesizes its own fatty acids. This work is the first to reveal the significance of balancing the making and taking of fatty acids in a Gram-negative bacterium during infection, which provides new insights into the validity of targeting fatty acid synthesis as a treatment strategy. IMPORTANCE Acinetobacter baumannii is one of the world's most problematic superbugs and is associated with significant morbidity and mortality in the hospital environment. The critical need for new antimicrobial strategies is recognized, but our understanding of its behavior and adaptation to a changing environment during infection is limited. Here, we investigated the role of fatty acids at the host-pathogen interface using a mouse model of disease. We provide comprehensive insights into the bacterial membrane composition when the bacteria colonize the pleural cavity. Furthermore, we show that A. baumannii heavily relies upon making its own fatty acids when residing in the blood, whereas the bacterium favors fatty acid acquisition in most other host niches. Our new knowledge aids in understanding the importance of host fatty acids in infectious diseases. Furthermore, fatty acid synthesis is an attractive target for the development of new antimicrobial strategies, but our work emphasizes the critical need to understand microbial lipid homeostasis before this can be deemed suitable.

Acinetobacter baumannii Fatty Acid Desaturases Facilitate Survival in Distinct Environments.

Maintaining optimal fluidity is essential to ensure adequate membrane structure and function under different environmental conditions. We apply integrated molecular approaches to characterize two desaturases (DesA and DesB) and define their specific roles in unsaturated fatty acid (UFA) production in Acinetobacter baumannii. Using a murine model, we reveal DesA to play a minor role in colonization of the respiratory tract, whereas DesB is important during invasive disease. Furthermore, using transcriptomic and bioinformatic analyses, a global regulator involved in fatty acid homeostasis and members of its regulon are characterized. Collectively, we show that DesA and DesB are primary contributors to UFA production in A. baumannii with infection studies illustrating that these distinct desaturases aid in the bacterium's ability to survive in multiple host niches. Hence, this study provides novel insights into the fundamentals of A. baumannii lipid biology, which contributes to the versatility of this critical bacterial pathogen.

Insights into Acinetobacter baumannii fatty acid synthesis 3-oxoacyl-ACP reductases.

Treatments for 'superbug' infections are the focus for innovative research, as drug resistance threatens human health and medical practices globally. In particular, Acinetobacter baumannii (Ab) infections are repeatedly reported as difficult to treat due to increasing antibiotic resistance. Therefore, there is increasing need to identify novel targets in the development of different antimicrobials. Of particular interest is fatty acid synthesis, vital for the formation of phospholipids, lipopolysaccharides/lipooligosaccharides, and lipoproteins of Gram-negative envelopes. The bacterial type II fatty acid synthesis (FASII) pathway is an attractive target for the development of inhibitors and is particularly favourable due to the differences from mammalian type I fatty acid synthesis. Discrete enzymes in this pathway include two reductase enzymes: 3-oxoacyl-acyl carrier protein (ACP) reductase (FabG) and enoyl-ACP reductase (FabI). Here, we investigate annotated FabG homologs, finding a low-molecular weight 3-oxoacyl-ACP reductase, as the most likely FASII FabG candidate, and high-molecular weight 3-oxoacyl-ACP reductase (HMwFabG), showing differences in structure and coenzyme preference. To date, this is the second bacterial high-molecular weight FabG structurally characterized, following FabG4 from Mycobacterium. We show that ΔAbHMwfabG is impaired for growth in nutrient rich media and pellicle formation. We also modelled a third 3-oxoacyl-ACP reductase, which we annotated as AbSDR. Despite containing residues for catalysis and the ACP coordinating motif, biochemical analyses showed limited activity against an acetoacetyl-CoA substrate in vitro. Inhibitors designed to target FabG proteins and thus prevent fatty acid synthesis may provide a platform for use against multidrug-resistant pathogens including A. baumannii.

The Role of Zinc Efflux during Acinetobacter baumannii Infection.

Acinetobacter baumannii is a ubiquitous Gram-negative bacterium, that is associated with significant disease in immunocompromised individuals. The success of A. baumannii is partly attributable to its high level of antibiotic resistance. Further, A. baumannii expresses a broad arsenal of putative zinc efflux systems that are likely to aid environmental persistence and host colonization, but detailed insights into how the bacterium deals with toxic concentrations of zinc are lacking. In this study we present the transcriptomic responses of A. baumannii to toxic zinc concentrations. Subsequent mutant analyses revealed a primary role for the resistance-nodulation-cell division heavy metal efflux system CzcCBA, and the cation diffusion facilitator transporter CzcD in zinc resistance. To examine the role of zinc at the host-pathogen interface we utilized a murine model of zinc deficiency and challenge with wild-type and czcA mutant strains, which identified highly site-specific roles for zinc during A. baumannii infection. Overall, we provide novel insight into the key zinc resistance mechanisms of A. baumannii and outline the role these systems play in enabling the bacterium to survive in diverse environments.

MITE Aba12 , a Novel Mobile Miniature Inverted-Repeat Transposable Element Identified in Acinetobacter baumannii ATCC 17978 and Its Prevalence across the Moraxellaceae Family.

Insertion sequences (IS) are fundamental mediators of genome plasticity with the potential to generate phenotypic variation with significant evolutionary outcomes. Here, a recently active miniature inverted-repeat transposon element (MITE) was identified in a derivative of Acinetobacter baumannii ATCC 17978 after being subjected to stress conditions. Transposition of the novel element led to the disruption of the hns gene, resulting in a characteristic hypermotile phenotype. DNA identity shared between the terminal inverted repeats of this MITE and coresident ISAba12 elements, together with the generation of 9-bp target site duplications, provides strong evidence that ISAba12 elements were responsible for mobilization of the MITE (designated MITE Aba12 ) within this strain. A wider genome-level survey identified MITE Aba12 in 30 additional Acinetobacter genomes at various frequencies and one Moraxella osloensis genome. Ninety MITE Aba12 copies could be identified, of which 40% had target site duplications, indicating recent transposition events. Elements ranged between 111 and 114 bp; 90% were 113 bp in length. Using the MITE Aba12 consensus sequence, putative outward-facing Escherichia coli σ70 promoter sequences in both orientations were identified. The identification of transcripts originating from the promoter in one direction supports the proposal that the element can influence neighboring host gene transcription. The location of MITE Aba12 varied significantly between and within genomes, preferentially integrating into AT-rich regions. Additionally, a copy of MITE Aba12 was identified in a novel 8.5-kb composite transposon, Tn6645, in the M. osloensis CCUG 350 chromosome. Overall, this study shows that MITE Aba12 is the most abundant nonautonomous element currently found in AcinetobacterIMPORTANCE One of the most important weapons in the armory of Acinetobacter is its impressive genetic plasticity, facilitating rapid genetic mutations and rearrangements as well as integration of foreign determinants carried by mobile genetic elements. Of these, IS are considered one of the key forces shaping bacterial genomes and ultimately evolution. We report the identification of a novel nonautonomous IS-derived element present in multiple bacterial species from the Moraxellaceae family and its recent translocation into the hns locus in the A. baumannii ATCC 17978 genome. The latter finding adds new knowledge to only a limited number of documented examples of MITEs in the literature and underscores the plastic nature of the hns locus in A. baumannii MITE Aba12 , and its predicted parent(s), may be a source of substantial adaptive evolution within environmental and clinically relevant bacterial pathogens and, thus, have broad implications for niche-specific adaptation.

Resistance to pentamidine is mediated by AdeAB, regulated by AdeRS, and influenced by growth conditions in Acinetobacter baumannii ATCC 17978.

In recent years, effective treatment of infections caused by Acinetobacter baumannii has become challenging due to the ability of the bacterium to acquire or up-regulate antimicrobial resistance determinants. Two component signal transduction systems are known to regulate expression of virulence factors including multidrug efflux pumps. Here, we investigated the role of the AdeRS two component signal transduction system in regulating the AdeAB efflux system, determined whether AdeA and/or AdeB can individually confer antimicrobial resistance, and explored the interplay between pentamidine resistance and growth conditions in A. baumannii ATCC 17978. Results identified that deletion of adeRS affected resistance towards chlorhexidine and 4',6-diamidino-2-phenylindole dihydrochloride, two previously defined AdeABC substrates, and also identified an 8-fold decrease in resistance to pentamidine. Examination of ΔadeA, ΔadeB and ΔadeAB cells augmented results seen for ΔadeRS and identified a set of dicationic AdeAB substrates. RNA-sequencing of ΔadeRS revealed transcription of 290 genes were ≥2-fold altered compared to the wildtype. Pentamidine shock significantly increased adeA expression in the wildtype, but decreased it in ΔadeRS, implying that AdeRS activates adeAB transcription in ATCC 17978. Investigation under multiple growth conditions, including the use of Biolog phenotypic microarrays, revealed resistance to pentamidine in ATCC 17978 and mutants could be altered by bioavailability of iron or utilization of different carbon sources. In conclusion, the results of this study provide evidence that AdeAB in ATCC 17978 can confer intrinsic resistance to a subset of dicationic compounds and in particular, resistance to pentamidine can be significantly altered depending on the growth conditions.

Diversity and Function of Capsular Polysaccharide in Acinetobacter baumannii.

The Gram-negative opportunistic bacterium Acinetobacter baumannii is a significant cause of hospital-borne infections worldwide. Alarmingly, the rapid development of antimicrobial resistance coupled with the remarkable ability of isolates to persist on surfaces for extended periods of time has led to infiltration of A. baumannii into our healthcare environments. A major virulence determinant of A. baumannii is the presence of a capsule that surrounds the bacterial surface. This capsule is comprised of tightly packed repeating polysaccharide units which forms a barrier around the bacterial cell wall, providing protection from environmental pressures including desiccation and disinfection regimes as well as host immune responses such as serum complement. Additionally, capsule has been shown to confer resistance to a range of clinically relevant antimicrobial compounds. Distressingly, treatment options for A. baumannii infections are becoming increasingly limited, and the urgency to develop effective infection control strategies and therapies to combat infections is apparent. An increased understanding of the contribution of capsule to the pathobiology of A. baumannii is required to determine its feasibility as a target for new strategies to combat drug resistant infections. Significant variation in capsular polysaccharide structures between A. baumannii isolates has been identified, with over 100 distinct capsule types, incorporating a vast variety of sugars. This review examines the studies undertaken to elucidate capsule diversity and advance our understanding of the role of capsule in A. baumannii pathogenesis.