The novel two-component system AmsSR governs alternative metabolic pathway usage in Acinetobacter baumannii.

In this study, we identify a novel two-component system in Acinetobacter baumannii (herein named AmsSR for regulator of alternative metabolic systems) only present in select gammaproteobacterial and betaproteobacterial species. Bioinformatic analysis revealed that the histidine kinase, AmsS, contains 14 predicted N-terminal transmembrane domains and harbors a hybrid histidine kinase arrangement in its C-terminus. Transcriptional analysis revealed the proton ionophore CCCP selectively induces P amsSR expression. Disruption of amsSR resulted in decreased intracellular pH and increased depolarization of cytoplasmic membranes. Transcriptome profiling revealed a major reordering of metabolic circuits upon amsR disruption, with energy generation pathways typically used by bacteria growing in limited oxygen being favored. Interestingly, we observed enhanced growth rates for mutant strains in the presence of glucose, which led to overproduction of pyruvate. To mitigate the toxic effects of carbon overflow, we noted acetate overproduction in amsSR-null strains, resulting from a hyperactive Pta-AckA pathway. Additionally, due to altered expression of key metabolic genes, amsSR mutants favor an incomplete TCA cycle, relying heavily on an overactive glyoxylate shunt. This metabolic reordering overproduces NADH, which is not oxidized by the ETC; components of which were significantly downregulated upon amsSR disruption. As a result, the mutants almost exclusively rely on substrate phosphorylation for ATP production, and consequently display reduced oxygen consumption in the presence of glucose. Collectively, our data suggests that disruption of amsSR affects the function of the aerobic respiratory chain, impacting the energy status of the cell, which in turn upregulates alternative metabolic and energy generation pathways.

c-di-AMP Is Essential for the Virulence of Enterococcus faecalis.

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (ΔcdaA strain) or c-di-AMP accumulation (ΔdhhP, ΔgdpP, and ΔdhhP ΔgdpP strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the ΔcdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo, and was virtually avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of the ΔcdaA strain also could be attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.

Regulatory networks important for survival of Acinetobacter baumannii within the host.

Acinetobacter baumannii is known for its intrinsic resistance to conventional antibiotic treatment and hypervirulence during infection. This coupled with its extraordinary capacity to survive in myriad harsh environments has led to increasing rates of infection in clinical settings. Numerous studies have characterized the virulence factors and resistance genes in A. baumannii responsible for the detrimental outcomes seen in patients; however, the role of regulatory factors in controlling the expression of these genes remains less well explored. Herein we discuss the latest and most influential findings on the regulatory network of A. baumannii, focusing on the transcription factors, two-component systems, and sRNAs. We place particular focus on those identified as being crucial for sensing and responding to continually changing environments, and influencing survival and virulence when engaging with the human host.

Genomes of ubiquitous marine and hypersaline Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira spp. encode a diversity of mechanisms to sustain chemolithoautotrophy in heterogeneous environments.

Chemolithoautotrophic bacteria from the genera Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira are common, sometimes dominant, isolates from sulfidic habitats including hydrothermal vents, soda and salt lakes and marine sediments. Their genome sequences confirm their membership in a deeply branching clade of the Gammaproteobacteria. Several adaptations to heterogeneous habitats are apparent. Their genomes include large numbers of genes for sensing and responding to their environment (EAL- and GGDEF-domain proteins and methyl-accepting chemotaxis proteins) despite their small sizes (2.1-3.1 Mbp). An array of sulfur-oxidizing complexes are encoded, likely to facilitate these organisms' use of multiple forms of reduced sulfur as electron donors. Hydrogenase genes are present in some taxa, including group 1d and 2b hydrogenases in Hydrogenovibrio marinus and H. thermophilus MA2-6, acquired via horizontal gene transfer. In addition to high-affinity cbb3 cytochrome c oxidase, some also encode cytochrome bd-type quinol oxidase or ba3 -type cytochrome c oxidase, which could facilitate growth under different oxygen tensions, or maintain redox balance. Carboxysome operons are present in most, with genes downstream encoding transporters from four evolutionarily distinct families, which may act with the carboxysomes to form CO2 concentrating mechanisms. These adaptations to habitat variability likely contribute to the cosmopolitan distribution of these organisms.

Towards the complete proteinaceous regulome of Acinetobacter baumannii.

The emergence of Acinetobacter baumannii strains, with broad multidrug-resistance phenotypes and novel virulence factors unique to hypervirulent strains, presents a major threat to human health worldwide. Although a number of studies have described virulence-affecting entities for this organism, very few have identified regulatory elements controlling their expression. Previously, our group has documented the global identification and curation of regulatory RNAs in A. baumannii. As such, in the present study, we detail an extension of this work, the performance of an extensive bioinformatic analysis to identify regulatory proteins in the recently annotated genome of the highly virulent AB5075 strain. In so doing, 243 transcription factors, 14 two-component systems (TCSs), 2 orphan response regulators, 1 hybrid TCS and 5 σ factors were found. A comparison of these elements between AB5075 and other clinical isolates, as well as a laboratory strain, led to the identification of several conserved regulatory elements, whilst at the same time uncovering regulators unique to hypervirulent strains. Lastly, by comparing regulatory elements compiled in this study to genes shown to be essential for AB5075 infection, we were able to highlight elements with a specific importance for pathogenic behaviour. Collectively, our work offers a unique insight into the regulatory network of A. baumannii strains, and provides insight into the evolution of hypervirulent lineages.

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

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

Unique structural modifications are present in the lipopolysaccharide from colistin-resistant strains of Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial opportunistic pathogen that can cause severe infections, including hospital-acquired pneumonia, wound infections, and sepsis. Multidrug-resistant (MDR) strains are prevalent, further complicating patient treatment. Due to the increase in MDR strains, the cationic antimicrobial peptide colistin has been used to treat A. baumannii infections. Colistin-resistant strains of A. baumannii with alterations to the lipid A component of lipopolysaccharide (LPS) have been reported; specifically, the lipid A structure was shown to be hepta-acylated with a phosphoethanolamine (pEtN) modification present on one of the terminal phosphate residues. Using a tandem mass spectrometry platform, we provide definitive evidence that the lipid A isolated from colistin-resistant A. baumannii MAC204 LPS contains a novel structure corresponding to a diphosphoryl hepta-acylated lipid A structure with both pEtN and galactosamine (GalN) modifications. To correlate our structural studies with clinically relevant samples, we characterized colistin-susceptible and -resistant isolates obtained from patients. These results demonstrated that the clinical colistin-resistant isolate had the same pEtN and GalN modifications as those seen in the laboratory-adapted A. baumannii strain MAC204. In summary, this work has shown complete structure characterization including the accurate assignment of acylation, phosphorylation, and glycosylation of lipid A from A. baumannii, which are important for resistance to colistin.

Activities of vancomycin-containing regimens against colistin-resistant Acinetobacter baumannii clinical strains.

Treatment of infections due to extensively drug-resistant (XDR) Acinetobacter baumannii often involves the use of antimicrobial agents in combination. Various combinations of agents have been proposed, with colistin serving as the backbone in many of them. Recent data suggest that glycopeptides, in particular vancomycin, may have unique activity against laboratory-adapted and clinical strains of A. baumannii, alone and in combination with colistin. The aim of the present study was to test this approach against three unique colistin-resistant A. baumannii clinical strains using combinations of vancomycin (VAN), colistin (COL), and doripenem (DOR). All three strains possessed the signature phosphoethanolamine modification of the lipid A moiety associated with colistin resistance and unique amino acid changes in the PmrAB two-component signal transduction system not observed in colistin-susceptible strains. In checkerboard assays, synergy (defined as a fractional inhibitory concentration index [FICI] of ≤ 0.5) was observed between COL and VAN for all three strains tested and between COL and DOR in two strains. In time-kill assays, the combinations of COL-DOR, COL-VAN, and COL-DOR-VAN resulted in complete killing of colistin-resistant A. baumannii in 1, 2, and all 3 strains, respectively. In the Galleria mellonella moth model of infection, the combinations of DOR-VAN and COL-DOR-VAN led to significantly increased survival of the larvae, compared with other combinations and monotherapy. These findings suggest that regimens containing vancomycin may confer therapeutic benefit for infection due to colistin-resistant A. baumannii.