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Human pleural fluid triggers global changes in the transcriptional landscape of Acinetobacter baumannii as an adaptive response to stress.

ABSTRACT: Acinetobacter baumannii is a feared, drug-resistant pathogen, characterized by its ability to resist extreme environmental and nutrient-deprived conditions. Previously, we showed that human serum albumin (HSA) can increase foreign DNA acquisition specifically and alter the expression of genes associated with pathogenicity. Moreover, in a recent genome-wide transcriptomic study, we observed that pleural fluid (PF), an HSA-containing fluid, increases DNA acquisition, can modulate cytotoxicity, and control immune responses by eliciting changes in the A. baumannii metabolic profile. In the present work, using more stringent criteria and focusing on the analysis of genes related to pathogenicity and response to stress, we analyzed our previous RNA-seq data and performed phenotypic assays to further explore the impact of PF on A. baumannii's microbial behavior and the strategies used to overcome environmental stress. We observed that PF triggered differential expression of genes associated with motility, efflux pumps, antimicrobial resistance, biofilm formation, two-component systems (TCSs), capsule synthesis, osmotic stress, and DNA-damage response, among other categories. Phenotypic assays of A. baumannii A118 and two other clinical A. baumannii strains, revealed differences in their responses to PF in motility, biofilm formation, antibiotic susceptibility, osmotic stress, and outer membrane vesicle (OMV) production, suggesting that these changes are strain specific. We conclude that A. baumannii's pathoadaptive responses is induced by HSA-containing fluids and must be part of this bacterium armamentarium to persist in hostile environments.

SUBMITTER: Martinez J

PROVIDER: S-EPMC6872806 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Human pleural fluid triggers global changes in the transcriptional landscape of Acinetobacter baumannii as an adaptive response to stress.

Martinez Jasmine J Fernandez Jennifer S JS Liu Christine C Hoard Amparo A Mendoza Anthony A Nakanouchi Jun J Rodman Nyah N Courville Robert R Tuttobene Marisel R MR Lopez Carolina C Gonzalez Lisandro J LJ Shahrestani Parvin P Papp-Wallace Krisztina M KM Vila Alejandro J AJ Tolmasky Marcelo E ME Bonomo Robert A RA Sieira Rodrigo R Ramirez Maria Soledad MS

Scientific reports 20191121 1

Acinetobacter baumannii is a feared, drug-resistant pathogen, characterized by its ability to resist extreme environmental and nutrient-deprived conditions. Previously, we showed that human serum albumin (HSA) can increase foreign DNA acquisition specifically and alter the expression of genes associated with pathogenicity. Moreover, in a recent genome-wide transcriptomic study, we observed that pleural fluid (PF), an HSA-containing fluid, increases DNA acquisition, can modulate cytotoxicity, and ...[more]

PMID: 31754169

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Project description:Analysis of the effect of human pleural fluid on the transcriptome of Acinetobacter baumanii A118 Acinetobacter baumannii (Ab) is one of the most treacherous pathogens among those causing hospital-acquired pneumonia (HAP). A. baumannii possesses an adaptable physiology, seen not only in its antibiotic resistance and virulence phenotypes, but also in its metabolic versatility. In this study, we observed that A. baumannii undergoes global transcriptional changes in response to human pleural fluid (PF), a key host-derived environmental signal. Differential gene expression analyses combined with experimental approaches revealed changes in A. baumannii metabolism, affecting cytotoxicity, persistence, bacterial killing and chemotaxis. Over 55% of the differentially expressed transcriptomic genes corresponded to metabolic processes, including the up regulation of glutamate, short chain fatty acid, and styrene metabolism. We observed an up regulation of the pyruvate dehydrogenase complex and found that pyruvate (PYR), in conjunction with PF, triggers an A. baumannii pathogenic behavior that adversely impacts human epithelial cell viability. Interestingly, PF also amplified A. baumannii cytotoxicity against murine macrophages, suggesting an immune evasion strategy implemented by A. baumannii. Moreover, we uncovered opposing metabolic strategies dependent on the degree of pathogenicity of the strains, where less pathogenic strains demonstrated greater utilization of PYR to promote persister formation in the presence of PF. Additionally, our transcriptomic analysis and growth studies of A. baumannii suggest the existence of an alternative phenylalanine (PA) catabolic route independent of the phenylacetic acid pathway, which converts PA to phenylpyruvate (PP) and shuttles intermediates into styrene metabolism. This alternative route promoted a neutrophil-evasive state, as PF-induced degradation of PP significantly reduced overall human neutrophil chemotaxis in ex vivo chemotactic assays. Taken together, these data highlights A. baumannii pathoadaptabililty in response to host signals and provide further insight into the role of bacterial metabolism in virulence traits, antibiotic persistence strategies, and host innate immune evasion.

Project description:Objectives: Carbapenem-resistant Acinetobacter baumannii (CRAB) are one of the most difficult pathogen to treat. The only drug recently approved by the FDA that is active against CRAB is cefiderocol. However, recent studies have shown higher all-cause mortality rate in the group of patients treated with cefiderocol, that may be due to heteroresistance, a phenotype characterized by the survival of a small proportion of cells in a population seemingly isogenic. Previous studies showed that adding human fluids to CRAB cultures can lead to CFDC heteroresistance. To better understand the nature of this phenomenon, we carried out molecular and phenotypic analyses of CRAB heteroresistant bacterial subpopulations. Methods: The CRAB strain AMA40 was cultured in the presence of cefiderocol and human pleural fluid (HPF) to isolate heteroresistant variants. Two of them, AMA40 IHC_1 and IHC_2, were subjected to whole genome sequencing and transcriptomic analysis to identify the mutations and transcriptomic changes responsible for the development of cefiderocol resistance. The impact of mutations on the pharmacodynamic activity of cefiderocol was assessed by susceptibility testing, EDTA and Boronic acid inhibition analysis, biofilm formation, and static time-kill assays. Results: Variants AMA40 IHC_1 and IHC_2 had 53 mutations, forty of which were common to both heteroresistant strains. None of the mutations are located inside genes associated with iron-uptake systems or β-lactam resistance. However, pipA, a gene associated with iron homeostasis in other species, was mutated in heteroresistant strains. Transcriptomic analyses revealed modifications in levels of expression of genes associated with antibiotic resistance. The blaNDM-1, blaADC-2, pbp3, and pbp1 were expressed at higher levels. At the same time, the carO and ompA genes’ expression was reduced. Collateral resistance to amikacin was observed in the heteroresistant variants. Static time-kill assays showed that when CA-MHB was supplemented with human serum albumin, the main protein component of HPF, cefiderocol killing activity was considerably reduced in all three strains. Conclusions: We conclude that heteroresistance to cefiderocol in CRAB, when exposed to fluids containing high HSA, is caused by mutations and modifications in the expression of genes associated with resistance to β-lactams.

Project description:Acinetobacter baumannii (Ab) is one of the most treacherous pathogens among those causing hospital-acquired pneumonia (HAP). A. baumannii possesses an adaptable physiology, seen not only in its antibiotic resistance and virulence phenotypes but also in its metabolic versatility. In this study, we observed that A. baumannii undergoes global transcriptional changes in response to human pleural fluid (PF), a key host-derived environmental signal. Differential gene expression analyses combined with experimental approaches revealed changes in A. baumannii metabolism, affecting cytotoxicity, persistence, bacterial killing, and chemotaxis. Over 1,220 genes representing 55% of the differentially expressed transcriptomic data corresponded to metabolic processes, including the upregulation of glutamate, short chain fatty acid, and styrene metabolism. We observed an upregulation by 1.83- and 2.61-fold of the pyruvate dehydrogenase complex subunits E3 and E2, respectively. We also found that pyruvate (PYR), in conjunction with PF, triggers an A. baumannii pathogenic behavior that adversely impacts human epithelial cell viability. Interestingly, PF also amplified A. baumannii cytotoxicity against murine macrophages, suggesting an immune evasion strategy implemented by A. baumannii. Moreover, we uncovered opposing metabolic strategies dependent on the degree of pathogenicity of the strains, where less pathogenic strains demonstrated greater utilization of PYR to promote persister formation in the presence of PF. Additionally, our transcriptomic analysis and growth studies of A. baumannii suggest the existence of an alternative phenylalanine (PA) catabolic route independent of the phenylacetic acid pathway, which converts PA to phenylpyruvate (PP) and shuttles intermediates into styrene metabolism. This alternative route promoted a neutrophil-evasive state, as PF-induced degradation of PP significantly reduced overall human neutrophil chemotaxis in ex vivo chemotactic assays. Taken together, these data highlight A. baumannii pathoadaptabililty in response to host signals and provide further insight into the role of bacterial metabolism in virulence traits, antibiotic persistence strategies, and host innate immune evasion.

Project description:IntroductionPleural tuberculosis (PlTB), the most common site of extrapulmonary TB, is characterized by a paucibacillary nature and a compartmentalized inflammatory response in the pleural cavity, both of which make diagnosis and management extremely challenging. Although transcriptional signatures for pulmonary TB have already been described, data obtained by using this approach for extrapulmonary tuberculosis and, specifically, for pleural tuberculosis are scarce and heterogeneous. In the present study, a set of candidate genes previously described in pulmonary TB was evaluated to identify and validate a transcriptional signature in clinical samples from a Brazilian cohort of PlTB patients and those with other exudative causes of pleural effusion.MethodsAs a first step, target genes were selected by a random forest algorithm with recursive feature elimination (RFE) from public microarray datasets. Then, peripheral blood (PB) and pleural fluid (PF) samples from recruited patients presenting exudative pleural effusion were collected during the thoracentesis procedure. Transcriptional analysis of the selected top 10 genes was performed by quantitative RT-PCR (RT-qPCR).ResultsReanalysis of the public datasets identified a set of candidate genes (CARD17, BHLHE40, FCGR1A, BATF2, STAT1, BTN3A1, ANKRD22, C1QB, GBP2, and SEPTIN4) that demonstrated a global accuracy of 89.5% in discriminating pulmonary TB cases from other respiratory diseases. Our validation cohort consisted of PlTB (n = 35) patients and non-TB (n = 34) ones. The gene expressions of CARD17, GBP2, and C1QB in PF at diagnosis were significantly different between the two (PlTB and non-TB) groups (p < 0.0001). It was observed that the gene expressions of CARD17 and GBP2 were higher in PlTB PF than in non-TB patients. C1QB showed the opposite behavior, being higher in the non-TB PF. After anti-TB therapy, however, GBP2 gene expression was significantly reduced in PlTB patients (p < 0.001). Finally, the accuracy of the three above-cited highlighted genes in the PF was analyzed, showing AUCs of 91%, 90%, and 85%, respectively. GBP2 was above 80% (sensitivity = 0.89/specificity = 0.81), and CARD17 showed significant specificity (Se = 0.69/Sp = 0.95) in its capacity to discriminate the groups.ConclusionCARD17, GBP2, and C1QB showed promise in discriminating PlTB from other causes of exudative pleural effusion by providing accurate diagnoses, thus accelerating the initiation of anti-TB therapy.

Project description:Infections caused by Carbapenem-resistant Acinetobacter baumannii (CRAB) isolates, such as hospital-acquired pneumonia (HAP), bacteremia, and skin and soft tissue infections, among others, are particularly challenging to treat. Cefiderocol, a chlorocatechol-substituted siderophore antibiotic, was approved by the U.S. Food and Drug Administration (FDA) in 2019 and prescribed for the treatment of CRAB infections. Despite the initial positive treatment outcomes with this antimicrobial, recent studies reported a higher-than-average all-cause mortality rate in patients treated with cefiderocol compared to the best available therapy. The cause(s) behind these outcomes remains unconfirmed. A plausible hypothesis is heteroresistance, a phenotype characterized by the survival of a small proportion of cells in a population that is seemingly isogenic. Recent results have demonstrated that the addition of human fluids to CRAB cultures leads to cefiderocol heteroresistance. Here, we describe the molecular and phenotypic analyses of CRAB heteroresistant bacterial subpopulations to better understand the nature of the less-than-expected successful outcomes after cefiderocol treatment. Isolation of heteroresistant variants of the CRAB strain AMA40 was carried out in cultures supplemented with cefiderocol and human pleural fluid (HPF). Two AMA40 variants, AMA40 IHC1 and IHC2, were resistant to cefiderocol. To identify mutations and gene expression changes associated with cefiderocol heteroresistance, we subjected these variants to whole genome sequencing and global transcriptional analysis. We then assessed the impact of these mutations on the pharmacodynamic activity of cefiderocol via susceptibility testing, EDTA and boronic acid inhibition analysis, biofilm formation, and static time-kill assays. Heteroresistant variants AMA40 IHC1 and AMA40 IHC2 have 53 chromosomal mutations, of which 40 are common to both strains. None of the mutations occurred in genes associated with high affinity iron-uptake systems or β-lactam resistance. However, transcriptional analyses demonstrated significant modifications in levels of expression of genes associated with iron-uptake systems or β-lactam resistance. The blaNDM-1 and blaADC-2, as well as various iron-uptake system genes, were expressed at higher levels than the parental strain. On the other hand, the carO and ompA genes' expression was reduced. One of the mutations common to both heteroresistant strains was mapped within ppiA, a gene associated with iron homeostasis in other species. Static time-kill assays demonstrated that supplementing cation-adjusted Mueller-Hinton broth with human serum albumin (HAS), the main protein component of HPF, considerably reduced cefiderocol killing activity for all three strains tested. Notably, collateral resistance to amikacin was observed in both variants. We conclude that exposing CRAB to fluids with high HSA concentrations facilitates the rise of heteroresistance associated with point mutations and transcriptional upregulation of genes coding for β-lactamases and biofilm formation. The findings from this study hold significant implications for understanding the emergence of CRAB resistance mechanisms against cefiderocol treatment. This understanding is vital for the development of treatment guidelines that can effectively address the challenges posed by CRAB infections.

Dataset Information

Human pleural fluid triggers global changes in the transcriptional landscape of Acinetobacter baumannii as an adaptive response to stress.

Publications

Human pleural fluid triggers global changes in the transcriptional landscape of Acinetobacter baumannii as an adaptive response to stress.

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