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

Unique Structural Modifications Are Present in the Lipopolysaccharide from Colistin-Resistant Strains of \u3ci\u3eAcinetobacter baumannii\u3c/i\u3e

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

Investigating the Proteinaceous Regulome of the \u3cem\u3eAcinetobacter baumannii\u3c/em\u3e

Acinetobacter baumannii is an opportunistic pathogen that overtime has evolved into one of the most problematic pathogens due to its ability to overcome antibiotic pressures and harshly environments in the host and hospital environments. In this context, its genomic evolution due to its capacity to acquire genes that contribute to its pathogenic and antibiotic resistance nature has been the subject of research in the last decades providing with the identification of several proteins aiding in the process of pathogenicity. Although these findings have contributed to our understanding of A. baumannii pathogenic traits, the regulatory network that control their expression are less understood. As such, our first efforts to study gene regulation in this organism were focused on defining the complete set of regulatory proteins in A. baumannii. As such, we examined the genome of a highly pathogenic multidrug resistance (MDR) A. baumannii strain AB5075 and comparative analyses to understand evolution of regulatory networks were performed using A. baumannii strains with different MDR profiles. This work generated a complete set of regulatory proteins identified in AB5075. This tool serves us as a foundation to investigate the function of uncharacterized regulatory proteins in this organism. As a result of this, we characterized the role of a two-component system in controlling metabolic pathways and found that its disruption is detrimental for energy generation processes. In addition, we defined the role of an extracytoplasmic sigma factor that is required for efficient growth in the presence of hemin as a sole iron source. Overall, the work of this dissertation presents findings describing regulatory roles of a novel TCS and a sigma factor that will extend the knowledge of gene regulation in A. baumannii

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

Towards the Complete Proteinaceous Regulome of Acinetobacter baumannii

Supplementary figures as publishedin Microbial Genomics in the following article: Towards the Complete Proteinaceous Regulome of Acinetobacter baumannii 10.1099/mgen.0.00010