A conserved zinc-binding site in Acinetobacter baumannii PBP2 required for elongasome-directed bacterial cell shape.

Acinetobacter baumannii is a gram-negative bacterial pathogen that causes challenging nosocomial infections. ß-lactam targeting of penicillin-binding protein (PBP)-mediated cell wall peptidoglycan (PG) formation is a well-established antimicrobial strategy. Exposure to carbapenems or zinc (Zn)-deprived growth conditions leads to a rod-to-sphere morphological transition in A. baumannii, an effect resembling that caused by deficiency in the RodA-PBP2 PG synthesis complex required for cell wall elongation. While it is recognized that carbapenems preferentially acylate PBP2 in A. baumannii and therefore block the transpeptidase function of the RodA-PBP2 system, the molecular details underpinning cell wall elongation inhibition upon Zn starvation remain undefined. Here, we report the X-ray crystal structure of A. baumannii PBP2, revealing an unexpected Zn coordination site in the transpeptidase domain required for protein stability. Mutations in the Zn-binding site of PBP2 cause a loss of bacterial rod shape and increase susceptibility to ß-lactams, therefore providing a direct rationale for cell wall shape maintenance and Zn homeostasis in A. baumannii. Furthermore, the Zn-coordinating residues are conserved in various ß- and γ-proteobacterial PBP2 orthologs, consistent with a widespread Zn-binding requirement for function that has been previously unknown. Due to the emergence of resistance to virtually all marketed antibiotic classes, alternative or complementary antimicrobial strategies need to be explored. These findings offer a perspective for dual inhibition of Zn-dependent PG synthases and metallo-ß-lactamases by metal chelating agents, considered the most sought-after adjuvants to restore ß-lactam potency against gram-negative bacteria.

Genome-wide phage susceptibility analysis in Acinetobacter baumannii reveals capsule modulation strategies that determine phage infectivity.

Phage have gained renewed interest as an adjunctive treatment for life-threatening infections with the resistant nosocomial pathogen Acinetobacter baumannii. Our understanding of how A. baumannii defends against phage remains limited, although this information could lead to improved antimicrobial therapies. To address this problem, we identified genome-wide determinants of phage susceptibility in A. baumannii using Tn-seq. These studies focused on the lytic phage Loki, which targets Acinetobacter by unknown mechanisms. We identified 41 candidate loci that increase susceptibility to Loki when disrupted, and 10 that decrease susceptibility. Combined with spontaneous resistance mapping, our results support the model that Loki uses the K3 capsule as an essential receptor, and that capsule modulation provides A. baumannii with strategies to control vulnerability to phage. A key center of this control is transcriptional regulation of capsule synthesis and phage virulence by the global regulator BfmRS. Mutations hyperactivating BfmRS simultaneously increase capsule levels, Loki adsorption, Loki replication, and host killing, while BfmRS-inactivating mutations have the opposite effect, reducing capsule and blocking Loki infection. We identified novel BfmRS-activating mutations, including knockouts of a T2 RNase protein and the disulfide formation enzyme DsbA, that hypersensitize bacteria to phage challenge. We further found that mutation of a glycosyltransferase known to alter capsule structure and bacterial virulence can also cause complete phage resistance. Finally, additional factors including lipooligosaccharide and Lon protease act independently of capsule modulation to interfere with Loki infection. This work demonstrates that regulatory and structural modulation of capsule, known to alter A. baumannii virulence, is also a major determinant of susceptibility to phage.

Acinetobacter baumannii: Envelope Determinants That Control Drug Resistance, Virulence, and Surface Variability.

Acinetobacter baumannii has emerged as an important nosocomial pathogen, particularly for patients in intensive care units and with invasive indwelling devices. The most recent clinical isolates are resistant to several classes of clinically important antibiotics, greatly restricting the ability to effectively treat critically ill patients. The bacterial envelope is an important driver of A. baumannii disease, both at the level of battling against antibiotic therapy and at the level of protecting from host innate immune function. This review provides a comprehensive overview of key features of the envelope that interface with both the host and antimicrobial therapies. Carbohydrate structures that contribute to protecting from the host are detailed, and mutations that alter these structures, resulting in increased antimicrobial resistance, are explored. In addition, protein complexes involved in both intermicrobial and host-microbe interactions are described. Finally we discuss regulatory mechanisms that control the nature of the cell envelope and its impact on host innate immune function.

Essential Gene Analysis in Acinetobacter baumannii by High-Density Transposon Mutagenesis and CRISPR Interference.

Acinetobacter baumannii is a poorly understood bacterium capable of life-threatening infections in hospitals. Few antibiotics remain effective against this highly resistant pathogen. Development of rationally designed antimicrobials that can target A. baumannii requires improved knowledge of the proteins that carry out essential processes allowing growth of the organism. Unfortunately, studying essential genes has been challenging using traditional techniques, which usually require time-consuming recombination-based genetic manipulations. Here, we performed saturating mutagenesis with dual transposon systems to identify essential genes in A. baumannii, and we developed a CRISPR interference (CRISPRi) system for facile analysis of these genes. We show that the CRISPRi system enables efficient transcriptional silencing in A. baumannii. Using these tools, we confirmed the essentiality of the novel cell division protein AdvA and discovered a previously uncharacterized AraC family transcription factor (ACX60_RS03245) that is necessary for growth. In addition, we show that capsule biosynthesis is a conditionally essential process, with mutations in late-acting steps causing toxicity in strain ATCC 17978 that can be bypassed by blocking early-acting steps or activating the BfmRS stress response. These results open new avenues for analysis of essential pathways in A. baumannii. IMPORTANCE New approaches are urgently needed to control A. baumannii, one of the most drug-resistant pathogens known. To facilitate the development of novel targets that allow inhibition of the pathogen, we performed a large-scale identification of genes whose products the bacterium needs for growth. We also developed a CRISPR-based gene knockdown tool that operates efficiently in A. baumannii, allowing rapid analysis of these essential genes. We used these methods to define multiple processes vital to the bacterium, including a previously uncharacterized gene regulatory factor and export of a protective polymeric capsule. These tools will enhance our ability to investigate processes critical for the essential biology of this challenging hospital-acquired pathogen.

A global regulatory system links virulence and antibiotic resistance to envelope homeostasis in Acinetobacter baumannii.

The nosocomial pathogen Acinetobacter baumannii is a significant threat due to its ability to cause infections refractory to a broad range of antibiotic treatments. We show here that a highly conserved sensory-transduction system, BfmRS, mediates the coordinate development of both enhanced virulence and resistance in this microorganism. Hyperactive alleles of BfmRS conferred increased protection from serum complement killing and allowed lethal systemic disease in mice. BfmRS also augmented resistance and tolerance against an expansive set of antibiotics, including dramatic protection from ß-lactam toxicity. Through transcriptome profiling, we showed that BfmRS governs these phenotypes through global transcriptional regulation of a post-exponential-phase-like program of gene expression, a key feature of which is modulation of envelope biogenesis and defense pathways. BfmRS activity defended against cell-wall lesions through both ß-lactamase-dependent and -independent mechanisms, with the latter being connected to control of lytic transglycosylase production and proper coordination of morphogenesis and division. In addition, hypersensitivity of bfmRS knockouts could be suppressed by unlinked mutations restoring a short, rod cell morphology, indicating that regulation of drug resistance, pathogenicity, and envelope morphogenesis are intimately linked by this central regulatory system in A. baumannii. This work demonstrates that BfmRS controls a global regulatory network coupling cellular physiology to the ability to cause invasive, drug-resistant infections.

Interplay Between Antibiotic Resistance and Virulence During Disease Promoted by Multidrug-Resistant Bacteria.

Diseases caused by antibiotic-resistant bacteria in hospitals are the outcome of complex relationships between several dynamic factors, including bacterial pathogenicity, the fitness costs of resistance in the human host, and selective forces resulting from interventions such as antibiotic therapy. The emergence and fate of mutations that drive antibiotic resistance are governed by these interactions. In this review, we will examine how different forms of antibiotic resistance modulate bacterial fitness and virulence potential, thus influencing the ability of pathogens to evolve in the context of nosocomial infections. We will focus on 3 important multidrug-resistant pathogens that are notoriously problematic in hospitals: Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus. An understanding of how antibiotic resistance mutations shape the pathobiology of multidrug-resistant infections has the potential to drive novel strategies that can control the development and spread of drug resistance.

Antibiotic modulation of capsular exopolysaccharide and virulence in Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen of increasing importance due to its propensity for intractable multidrug-resistant infections in hospitals. All clinical isolates examined contain a conserved gene cluster, the K locus, which determines the production of complex polysaccharides, including an exopolysaccharide capsule known to protect against killing by host serum and to increase virulence in animal models of infection. Whether the polysaccharides determined by the K locus contribute to intrinsic defenses against antibiotics is unknown. We demonstrate here that mutants deficient in the exopolysaccharide capsule have lowered intrinsic resistance to peptide antibiotics, while a mutation affecting sugar precursors involved in both capsule and lipopolysaccharide synthesis sensitizes the bacterium to multiple antibiotic classes. We observed that, when grown in the presence of certain antibiotics below their MIC, including the translation inhibitors chloramphenicol and erythromycin, A. baumannii increases production of the K locus exopolysaccharide. Hyperproduction of capsular exopolysaccharide is reversible and non-mutational, and occurs concomitantly with increased resistance to the inducing antibiotic that is independent of the presence of the K locus. Strikingly, antibiotic-enhanced capsular exopolysaccharide production confers increased resistance to killing by host complement and increases virulence in a mouse model of systemic infection. Finally, we show that augmented capsule production upon antibiotic exposure is facilitated by transcriptional increases in K locus gene expression that are dependent on a two-component regulatory system, bfmRS. These studies reveal that the synthesis of capsule, a major pathogenicity determinant, is regulated in response to antibiotic stress. Our data are consistent with a model in which gene expression changes triggered by ineffectual antibiotic treatment cause A. baumannii to transition between states of low and high virulence potential, which may contribute to the opportunistic nature of the pathogen.

Identification of essential genes that support fitness of Acinetobacter baumannii efflux pump overproducers in the presence of fluoroquinolone.

Acinetobacter baumannii is a nosocomial pathogen often associated with multidrug resistance (MDR) infections. Fluoroquinolone resistance (FQR) due to drug target site mutations and elevated expression of RND drug transporters is common among clinical isolates. We describe here a CRISPRi platform that identifies hypomorphic mutations that preferentially altered drug sensitivity in RND pump overproducers. An sgRNA library against essential genes of A. baumannii was constructed with single and double nucleotide mutations that produced titratable knockdown efficiencies and introduced into multiple strain backgrounds. Other than nusG depletions, there were few candidates in the absence of drug treatment that showed lowered fitness specifically in strains overexpressing clinically relevant RND efflux pumps AdeAB, AdeIJK, or AdeFGH. In the presence of ciprofloxacin, the hypomorphs causing hypersensitivity were predicted to result in outer membrane dysfunction, to which the AdeFGH overproducer appeared particularly sensitive. Depletions of either the outer membrane assembly BAM complex, LOS biogenesis proteins, or Lpt proteins involved in LOS transport to the outer membrane caused drug hypersensitivity in at least two of the three pump overproducers. On the other hand, depletions of translation-associated proteins, as well as components of the proton-pumping ATP synthase pump resulted in fitness benefits for at least two pump-overproducing strains in the presence of the drug. Therefore, pump overproduction exacerbated stress caused by defective outer membrane integrity, while the efficacy of drug resistance in efflux overproducers was enhanced by slowed translation or defects in ATP synthesis linked to the control of proton movement across the bacterial membrane.

A phosphorylation signal activates genome-wide transcriptional control by BfmR, the global regulator of Acinetobacter resistance and virulence.

The nosocomial pathogen Acinetobacter baumannii is a major threat to human health. The sensor kinase-response regulator system, BfmS-BfmR, is essential to multidrug resistance and virulence in the bacterium and represents a potential antimicrobial target. Important questions remain about how the system controls resistance and pathogenesis. Although BfmR knockout alters expression of >1000 genes, its direct regulon is undefined. Moreover, how phosphorylation controls the regulator is unclear. Here, we address these problems by combining mutagenesis, ChIP-seq, and in vitro phosphorylation to study the functions of phospho-BfmR. We show that phosphorylation is required for BfmR-mediated gene regulation, antibiotic resistance, and sepsis development in vivo. Consistent with activating the protein, phosphorylation induces dimerization and target DNA affinity. Integrated analysis of genome-wide binding and transcriptional profiles of BfmR led to additional key findings: (1) Phosphorylation dramatically expands the number of genomic sites BfmR binds; (2) DNA recognition involves a direct repeat motif widespread across promoters; (3) BfmR directly regulates 303 genes as activator (eg, capsule, peptidoglycan, and outer membrane biogenesis) or repressor (pilus biogenesis); (4) BfmR controls several non-coding sRNAs. These studies reveal the centrality of a phosphorylation signal in driving A. baumannii disease and disentangle the extensive pathogenic gene-regulatory network under its control.

Identification of Determinants that Allow Maintenance of High-Level Fluoroquinolone Resistance in Acinetobacter baumannii.

Acinetobacter baumannii is associated with multidrug resistant (MDR) infections in healthcare settings, with fluoroquinolones such as ciprofloxacin being currently ineffective. Clinical isolates largely harbor mutations in the GyrA and TopoIV fluoroquinolone targets, as well as mutations that increase expression of drug resistance-nodulation-division (RND) efflux pumps. Factors critical for maintaining fitness levels of pump overproducers are uncharacterized despite their prevalence in clinical isolates. We here identify proteins that contribute to the fitness of FQR strains overexpressing three known RND systems using high-density insertion mutagenesis. Overproduction of the AdeFGH efflux pump caused hypersensitization to defects in outer membrane homeostatic regulation, including lesions that reduced LOS biosynthesis and blocked production of the major A. baumannii porin. In contrast, AdeAB pump overproduction, which does not affect the outer membrane pump component, was relatively tolerant to loss of these functions, consistent with outer membrane protein overproduction being the primary disruptive component. Surprisingly, overproduction of proton-transporting efflux pumps had little impact on cytosolic pH, consistent with a compensatory response to pump activity. The most striking transcriptional changes were associated with AdeFGH pump overproduction, resulting in activation of the phenylacetate (PAA) degradation regulon. Disruption of the PAA pathway resulted in cytosolic acidification and defective expression of genes involved in protection from peroxide stress. These results indicate that the RND outer membrane protein overproduction is compensated by cytoplasmic buffering and maintenance of outer membrane integrity in A. baumannii to facilitate fitness of FQR isolates.