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

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

Increased expression of <i>adeA</i> following pentamidine stress is dependent on the presence of AdeRS in ATCC 17978.

<p>Transcriptional levels of <i>adeA</i> (ACX60_09125) from WT and Δ<i>adeRS</i> were determined by qRT-PCR after 30 min shock with 7.8 mg/L of pentamidine (Pent) (0.5 × MIC of Δ<i>adeRS</i>) and corrected to untreated cells (UT) after normalization to 16S. Bars represent the mean fold change (Log₂) of three biological replicates undertaken in triplicate, and error bars represent ± SEM. Statistical analyses were performed by Student’s <i>t</i>-test, two-tailed, unpaired; ** = <i>P</i> < 0.01 and *** = <i>P</i> < 0.001.</p

Kinetic response curves paralleling bacterial growth from Biolog PM01 and PM2A plates identify ten carbon sources that increase pentamidine resistance in Δ<i>adeRS</i>.

<p>P8, P16, P32 and P64 represent kinetic response curves at 8, 16, 32, or 64 mg/L of pentamidine compared to the untreated control, respectively. Red curves represent respiration of untreated Δ<i>adeRS</i>, whilst respiratory activity which overlaps between the control and the sample in the different experimental conditions is represented in yellow. Only carbon compounds that promote at least 50% maximal respiration and induce a recovery response by 36 h are shown. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197412#pone.0197412.s003" target="_blank">S3 Fig</a> for respiration curves for all tested treatments.</p

Global transcriptomic response differences of <i>A</i>. <i>baumannii</i> ATCC 17978 after deletion of <i>adeRS</i>.

<p>Each diamond marker represents a predicted gene within the genome ordered according to locus tag along the X-axis and differential expression generated from normalized reads per kilobase mapped of WT against Δ<i>adeRS</i> are displayed on the Y-axis (Log₂). Positive and negative Log₂-values correlate to up- and down-regulated genes, respectively. Green and red circles highlight genes/gene clusters of interest that have been up- and down-regulated, respectively. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197412#pone.0197412.s007" target="_blank">S3 Table</a> for the full list of genes that were differentially expressed ≥ 1 Log₂ fold.</p

Resistance to pentamidine is modulated by carbon sources available in the growth medium.

<p>Ten-fold serial dilutions of <i>A</i>. <i>baumannii</i> ATCC 17978 (WT), Δ<i>adeRS</i> (Δ<i>RS</i>) and Δ<i>adeAB</i> (Δ<i>AB</i>) cells were used to compare the concentration of pentamidine that inhibits growth in different media, Mueller-Hinton agar (MHA) was used as a comparative control. Images display serial 1:10 dilutions after overnight incubation at 37°C, where DF is abbreviated for dilution factor and N represents undiluted cells. Strains were grown in the absence of pentamidine (UT) or presence of 32, 64, 128, 256 and 512 mg/L of pentamidine (P32, P64, P128, P256, and P512, respectively). Carbon sources tested in M9 minimal medium were used at a final concentration of 0.4% (w/v). ND, not done due to precipitation of pentamidine once added into the molten medium. Figures are representative examples of results obtained.</p

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

Comparative sequence, structure and redox analyses of Klebsiella pneumoniae DsbA show that anti-virulence target DsbA enzymes fall into distinct classes

Bacterial DsbA enzymes catalyze oxidative folding of virulence factors, and have been identified as targets for antivirulence drugs. However, DsbA enzymes characterized to date exhibit a wide spectrum of redox properties and divergent structural features compared to the prototypical DsbA enzyme of Escherichia coli DsbA (EcDsbA). Nonetheless, sequence analysis shows that DsbAs are more highly conserved than their known substrate virulence factors, highlighting the potential to inhibit virulence across a range of organisms by targeting DsbA. For example, Salmonella enterica typhimurium (SeDsbA, 86 % sequence identity to EcDsbA) shares almost identical structural, surface and redox properties. Using comparative sequence and structure analysis we predicted that five other bacterial DsbAs would share these properties. To confirm this, we characterized Klebsiella pneumoniae DsbA (KpDsbA, 81 % identity to EcDsbA). As expected, the redox properties, structure and surface features (from crystal and NMR data) of KpDsbA were almost identical to those of EcDsbA and SeDsbA. Moreover, KpDsbA and EcDsbA bind peptides derived from their respective DsbBs with almost equal affinity, supporting the notion that compounds designed to inhibit EcDsbA will also inhibit KpDsbA. Taken together, our data show that DsbAs fall into different classes; that DsbAs within a class may be predicted by sequence analysis of binding loops; that DsbAs within a class are able to complement one another in vivo and that compounds designed to inhibit EcDsbA are likely to inhibit DsbAs within the same class