Contribution of the a-baumannii A1S_0114 gene to the interaction with eukaryotic cells and virulence

Genetic and functional studies showed that some components of the Acinetobacter baumannii ATCC 17978 A1S_0112-A1S_0119 gene cluster are critical for biofilm biogenesis and surface motility. Recently, our group has shown that the A1S_0114 gene was involved in biofilm formation, a process related with pathogenesis. Confirming our previous results, microscopy images revealed that the ATCC 17978 10114 derivative lacking this gene was unable to form a mature biofilm structure. Therefore, other bacterial phenotypes were analyzed to determine the role of this gene in the pathogenicity of A. baumannii ATCC 17978. The interaction of the ATCC 17978 parental strain and the 10114 mutant with A549 human alveolar epithelial cells was quantified revealing that the A1S_0114 gene was necessary for proper attachment to A549 cells. This dependency correlates with the negative effect of the A1S_0114 deletion on the expression of genes coding for surface proteins and pili-assembly systems, which are known to play a role in adhesion. Three different experimental animal models, including vertebrate and invertebrate hosts, confirmed the role of the A1S_0114 gene in virulence. All of the experimental infection assays indicated that the virulence of the ATCC 17978 was significantly reduced when this gene was inactivated. Finally, we discovered that the A1S_0114 gene was involved in the production of a small lipopeptide-like compound herein referred to as acinetin 505 (Ac-505). Ac-505 was isolated from ATCC 17978 spent media and its chemical structure was interpreted by mass spectrometry. Overall, our observations provide novel information on the role of the A1S_0114 gene in A. baumannii’s pathobiology and lay the foundation for future work to determine the mechanisms by which Ac-505, or possibly an Ac-505 precursor, could execute critical functions as a secondary metaboliteS

Two Acinetobacter baumannii Isolates Obtained From a Fatal Necrotizing Fasciitis Infection Display Distinct Genomic and Phenotypic Characteristics in Comparison to Type Strains

Acinetobacter baumannii has been recognized as a critical pathogen that causes severe infections worldwide not only because of the emergence of extensively drug-resistant (XDR) derivatives, but also because of its ability to persist in medical environments and colonize compromised patients. While there are numerous reports describing the mechanisms by which this pathogen acquires resistance genes, little is known regarding A. baumannii's virulence functions associated with rare manifestations of infection such as necrotizing fasciitis, making the determination and implementation of alternative therapeutic targets problematic. To address this knowledge gap, this report describes the analysis of the NFAb-1 and NFAb-2 XDR isolates, which were obtained at two time points during a fatal case of necrotizing fasciitis, at the genomic and functional levels. The comparative genomic analysis of these isolates with the ATCC 19606T and ATCC 17978 strains showed that the NFAb-1 and NFAb-2 isolates are genetically different from each other as well as different from the ATCC 19606T and ATCC 17978 clinical isolates. These genomic differences could be reflected in phenotypic differences observed in these NFAb isolates. Biofilm, cell viability and flow cytometry assays indicate that all tested strains caused significant decreases in A549 human alveolar epithelial cell viability with ATCC 17978, NFAb-1 and NFAb-2 producing significantly less biofilm and significantly more hemolysis and capacity for intracellular invasion than ATCC 19606T. NFAb-1 and NFAb-2 also demonstrated negligible surface motility but significant twitching motility compared to ATCC 19606T and ATCC 17978, likely due to the presence of pili exceeding 2 µm in length, which are significantly longer and different from those previously described in the ATCC 19606T and ATCC 17978 strains. Interestingly, infection with cells of the NFAb-1 isolate, which were obtained from a premortem blood sample, lead to significantly higher mortality rates than NFAb-2 bacteria, which were obtained from postmortem tissue samples, when tested using the Galleria mellonella in vivo infection model. These observations suggest potential changes in the virulence phenotype of the A. baumannii necrotizing fasciitis isolates over the course of infection by mechanisms and cell processes that remain to be identified

Antimicrobial Activity of Gallium Protoporphyrin IX against Acinetobacter baumannii Strains Displaying Different Antibiotic Resistance Phenotypes

A paucity of effective, currently available antibiotics and a lull in antibiotic development pose significant challenges for treatment of patients with multidrug-resistant (MDR) Acinetobacter baumannii infections. Thus, novel therapeutic strategies must be evaluated to meet the demands of treatment of these often life-threatening infections. Accordingly, we examined the antibiotic activity of gallium protoporphyrin IX (Ga-PPIX) against a collection of A. baumannii strains, including nonmilitary and military strains and strains representing different clonal lineages and isolates classified as susceptible or MDR. Susceptibility testing demonstrated that Ga-PPIX inhibits the growth of all tested strains when cultured in cation-adjusted Mueller-Hinton broth, with a MIC of 20 μg/ml. This concentration significantly reduced bacterial viability, while 40 μg/ml killed all cells of the A. baumannii ATCC 19606(T) and ACICU MDR isolate after 24-h incubation. Recovery of ATCC 19606(T) and ACICU strains from infected A549 human alveolar epithelial monolayers was also decreased when the medium was supplemented with Ga-PPIX, particularly at a 40-μg/ml concentration. Similarly, the coinjection of bacteria with Ga-PPIX increased the survival of Galleria mellonella larvae infected with ATCC 19606(T) or ACICU. Ga-PPIX was cytotoxic only when monolayers or larvae were exposed to concentrations 16-fold and 1,250-fold higher than those showing antibacterial activity, respectively. These results indicate that Ga-PPIX could be a viable therapeutic option for treatment of recalcitrant A. baumannii infections regardless of the resistance phenotype, clone lineage, time and site of isolation of strains causing these infections and their iron uptake phenotypes or the iron content of the media

Differential transcription of genes coding for benzoate transport and metabolism functions in response to the presence of mucin.

<p>(A) A1S_1215-A1S_1206 genomic region coding for BenA/B/C/D/E/K/P orthologs. The arrows represent each coding region and its direction of transcription. The grey arrows represent genes up-regulated in response to the presence of mucin. The black arrow indicates the gene (<i>benP</i>, A1S_1209) that was used to confirm the mucin-mediated up-regulation effect by qRT-PCR. Numbers above the arrows represent cognate A1S_ gene annotation numbers, and gene names are indicated below each arrow. (B) qRT-PCR analysis of the differential expression of A1S_1209 in bacterial cells cultured in SB (white bar) or SB+M (grey bar) performed using nine replicates. Significantly different values (<i>P</i> ≤ 0.001) are identified by three asterisks and error bars represent the standard error of each data set.</p

<i>A</i>. <i>baumannii</i> ATCC 19606^T ion metabolism/transport-associated genes with decreased expression in the presence of mucin.

<p><i>A</i>. <i>baumannii</i> ATCC 19606^T ion metabolism/transport-associated genes with decreased expression in the presence of mucin.</p

<i>A</i>. <i>baumannii</i> ATCC 19606^T metabolic genes with increased expression in the presence of mucin.

<p><i>A</i>. <i>baumannii</i> ATCC 19606^T metabolic genes with increased expression in the presence of mucin.</p

<i>A</i>. <i>baumannii</i> ATCC 19606^T virulence-associated genes with increased expression in the presence of mucin.

<p><i>A</i>. <i>baumannii</i> ATCC 19606^T virulence-associated genes with increased expression in the presence of mucin.</p

Differential expression of <i>bauA</i> in response to the presence of mucin.

<p>(A) qRT-PCR analysis of <i>bauA</i> in bacterial cells cultured in SB (white bar) or SB+M (grey bar) performed using nine replicates. Significantly different values (<i>P</i> ≤ 0.0001) are identified by four asterisks and error bars represent the standard error of each data set. (B) Western blot of whole-cell lysate proteins obtained from ATCC 19606^T bacterial cells grown in LB (LB), LB supplemented with 100 μM dipyridyl (LB+D) or 100 μM FeCl₃ (LB+Fe), SB, or SB+M. E, empty lane. SDS-PAGE size-fractionated proteins were blotted onto nitrocellulose and probed with anti-BauA polyclonal antibodies. The western blot shown in this panel is a representative image of three blots obtained with three independent biological samples prepared as described above. All three blots produced the same outcome.</p

Effect of mucin on the expression of the <i>csuAB</i> gene, biofilm formation on plastic and pili production.

<p>(A) qRT-PCR analysis of <i>csuAB</i> in ATCC 19606^T cells cultured in SB (white bars) or SB+M (grey bars). (B) Crystal violet staining of biofilms formed by ATCC 19606^T on uncoated (U, white bar) or mucin-coated (M, grey bar) polystyrene tubes. qRT-PCR and biofilm analyses were performed using nine and six replicates for each study, respectively. Significantly different values (<i>P</i> ≤ 0.0001) are identified by four asterisks and error bars represent the standard error of each data set. (C) SEM of biofilms formed on uncoated (a, b, c) or mucin coated (d, e, f) coverslips by ATCC 19606^T cells. Micrographs were taken above (a and d), at (b and e), or below (c and f) the liquid-air interface at a magnification of 10,000x. Scale bars, 1 μm. (D) TEM of bacterial cells lifted from the surface of SA (g) or SA supplemented with mucin (h). Micrographs were taken at a magnification of 30,000x. Scale bars, 0.5 μm.</p

Mucin acts as a nutrient source and a signal for the differential expression of genes coding for cellular processes and virulence factors in <i>Acinetobacter baumannii</i>

<div><p>The capacity of <i>Acinetobacter baumannii</i> to persist and cause infections depends on its interaction with abiotic and biotic surfaces, including those found on medical devices and host mucosal surfaces. However, the extracellular stimuli affecting these interactions are poorly understood. Based on our previous observations, we hypothesized that mucin, a glycoprotein secreted by lung epithelial cells, particularly during respiratory infections, significantly alters <i>A</i>. <i>baumannii</i>’s physiology and its interaction with the surrounding environment. Biofilm, virulence and growth assays showed that mucin enhances the interaction of <i>A</i>. <i>baumannii</i> ATCC 19606^T with abiotic and biotic surfaces and its cytolytic activity against epithelial cells while serving as a nutrient source. The global effect of mucin on the physiology and virulence of this pathogen is supported by RNA-Seq data showing that its presence in a low nutrient medium results in the differential transcription of 427 predicted protein-coding genes. The reduced expression of ion acquisition genes and the increased transcription of genes coding for energy production together with the detection of mucin degradation indicate that this host glycoprotein is a nutrient source. The increased expression of genes coding for adherence and biofilm biogenesis on abiotic and biotic surfaces, the degradation of phenylacetic acid and the production of an active type VI secretion system further supports the role mucin plays in virulence. Taken together, our observations indicate that <i>A</i>. <i>baumannii</i> recognizes mucin as an environmental signal, which triggers a response cascade that allows this pathogen to acquire critical nutrients and promotes host-pathogen interactions that play a role in the pathogenesis of bacterial infections.</p></div