Abstract
Acinetobacter baumannii is an opportunistic nosocomial pathogen that is the causative agent of several serious infections in humans, including pneumonia, sepsis, and wound and burn infections. A. baumannii is also capable of forming proteinaceous biofilms on both abiotic and epithelial cell surfaces. Here, we investigate the response of A. baumannii toward sodium sulfide (Na2S), known to be associated with some biofilms at oxic/anoxic interfaces. The addition of exogenous inorganic sulfide reveals that A. baumannii encodes two persulfide-sensing transcriptional regulators, a primary σ54-dependent transcriptional activator (FisR), and a secondary system controlled by the persulfide-sensing biofilm growth-associated repressor (BigR), which is only induced by sulfide in a fisR deletion strain. FisR activates an operon encoding a sulfide oxidation/detoxification system similar to that characterized previously in Staphylococcus aureus, while BigR regulates a secondary persulfide dioxygenase (PDO2) as part of yeeE-yedE-pdo2 sulfur detoxification operon, found previously in Serratia spp. Global S-sulfuration (persulfidation) mapping of the soluble proteome reveals 513 persulfidation targets well beyond FisR-regulated genes and includes five transcriptional regulators, most notably the master biofilm regulator BfmR and a poorly characterized catabolite regulatory protein (Crp). Both BfmR and Crp are well known to impact biofilm formation in A. baumannii and other organisms, respectively, suggesting that persulfidation of these regulators may control their activities. The implications of these findings on bacterial sulfide homeostasis, persulfide signaling, and biofilm formation are discussed.IMPORTANCE Although hydrogen sulfide (H2S) has long been known as a respiratory poison, recent reports in numerous bacterial pathogens reveal that H2S and more downstream oxidized forms of sulfur collectedly termed reactive sulfur species (RSS) function as antioxidants to combat host efforts to clear the infection. Here, we present a comprehensive analysis of the transcriptional and proteomic response of A. baumannii to exogenous sulfide as a model for how this important human pathogen manages sulfide/RSS homeostasis. We show that A. baumannii is unique in that it encodes two independent persulfide sensing and detoxification pathways that govern the speciation of bioactive sulfur in cells. The secondary persulfide sensor, BigR, impacts the expression of biofilm-associated genes; in addition, we identify two other transcriptional regulators known or projected to regulate biofilm formation, BfmR and Crp, as highly persulfidated in sulfide-exposed cells. These findings significantly strengthen the connection between sulfide homeostasis and biofilm formation in an important human pathogen.
Highlights
IMPORTANCE hydrogen sulfide (H2S) has long been known as a respiratory poison, recent reports in numerous bacterial pathogens reveal that H2S and more downstream oxidized forms of sulfur collectedly termed reactive sulfur species (RSS) function as antioxidants to combat host efforts to clear the infection
The discovery and characterization of organic persulfide (RSS)-sensing transcriptional regulators, including CstR, SqrR/biofilm growth-associated repressor (BigR), and FisR, that regulate the expression of enzymes that are known or projected to reduce the cellular load of H2S and organic RSS led us to propose the concept of bacterial sulfide or RSS homeostasis [22,23,24,25,26]
In order to understand the regulatory response of A. baumannii ATCC 17978 to external sulfide stress, we used this approach to quantify endogenous levels of H2S, major cellular thiols and thiol persulfides in mid-log-phase wild-type (WT) cells and compare these levels to those in a strain lacking the 3-mercaptopyruvate sulfurtransferase (3MST) (A1S_3379). 3MST is thought to be an important source of endogenously synthesized H2S from studies of E. coli [5, 6, 8]
Summary
IMPORTANCE hydrogen sulfide (H2S) has long been known as a respiratory poison, recent reports in numerous bacterial pathogens reveal that H2S and more downstream oxidized forms of sulfur collectedly termed reactive sulfur species (RSS) function as antioxidants to combat host efforts to clear the infection. Low levels of H2S enhance the respiration, energy production, and survival of Mycobacterium tuberculosis in infected mice [9] These findings suggest that the maintenance of endogenous H2S reduces the efficacy of first-line antibiotics and cellular redox balance in infected animals, consistent with other studies in clinically isolated multidrug-resistant UPEC strains [10] and in S. aureus [11]. These data support the proposal that H2S functions as an infection-relevant antioxidant, which may be attributed to more downstream oxidized reactive sulfur species (RSS) [12]. Protein S-sulfuration (persulfidation) profiling in A. baumannii reveals that ϳ13% of the proteome is persulfidated and identifies potential regulatory targets of H2S/RSS stress, including two highly persulfidated transcriptional regulators, BfmR and Crp, known or projected to function in biofilm regulation
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