The Response of Acinetobacter baumannii to Hydrogen Sulfide Reveals Two Independent Persulfide-Sensing Systems and a Connection to Biofilm Regulation

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.

H2S and reactive sulfur signaling at the host-bacterial pathogen interface

“…Although cytochrome bd oxidase does not pump protons, it still enables aerobic metabolism and robust growth. This respiratory switch in response to H 2 S may well occur in several other bacterial pathogens, including Acinetobacter baumannii (20) and Mycobacterium tuberculosis; in the latter case, low levels of H 2 S enhance the respiration, energy production, and survival of M. tuberculosis in infected mice (25). Further studies are required to establish the generality of this adaptive response across a wider range of organisms.…”

“…It was not until 2014 that RSS were shown to function as antioxidants in mammalian cells (47), which may partly explain H 2 S-enhanced bacterial resistance to antibiotics (21). RSS have been detected in a number of bacterial pathogens (17,20,53,96) and are the subject of ongoing work to better understand the role of RSS in the bacterial response to the host immune system.…”

“…1A, top) (11)(12)(13)(14). In an analogous fashion, bacteria encode specialized transcriptional regulators that sense oxidized or "reactive" sulfur species (RSS), derived from hydrogen sulfide (H 2 S) (15)(16)(17)(18)(19)(20). As cellular concentrations of RSS rise, RSS sensors turn on the expression of genes that encode enzymes that reduce cellular loads of H 2 S/RSS to avoid H 2 S toxicity and overpersulfidation of the metabolome and proteome (Fig.…”

“…1B, bottom) (27). Analogous to a metallosensor, known RSS sensors specifically surveil the cytoplasm for a particular feature of the RSS pool, in this case sulfane sulfur (15)(16)(17)(18)(19)(20).…”

“…These dual sensors collaboratively control metal bioavailability in a concentration range that is compatible with cellular physiology (gray box). H 2 S/RSS homeostasis (bottom) is achieved by a single RSS sensor that transcriptionally regulates the expression of enzymes involved in the biogenesis, clearance, transport, and assimilation of H 2 S/RSS (16)(17)(18)(19)(20). The transcriptional response of an RSS sensor detects a concentration range (gray box) that prevents cellular toxicity, while maintaining access to H 2 S/RSS that is physiologically beneficial at lower concentrations.…”