A genome-wide screen in macrophages identifies PTEN as required for myeloid restriction of Listeria monocytogenes infection.

Listeria monocytogenes (Lm) is an intracellular foodborne pathogen which causes the severe disease listeriosis in immunocompromised individuals. Macrophages play a dual role during Lm infection by both promoting dissemination of Lm from the gastrointestinal tract and limiting bacterial growth upon immune activation. Despite the relevance of macrophages to Lm infection, the mechanisms underlying phagocytosis of Lm by macrophages are not well understood. To identify host factors important for Lm infection of macrophages, we performed an unbiased CRISPR/Cas9 screen which revealed pathways that are specific to phagocytosis of Lm and those that are required for internalization of bacteria generally. Specifically, we discovered the tumor suppressor PTEN promotes macrophage phagocytosis of Lm and L. ivanovii, but not other Gram-positive bacteria. Additionally, we found that PTEN enhances phagocytosis of Lm via its lipid phosphatase activity by promoting adherence to macrophages. Using conditional knockout mice lacking Pten in myeloid cells, we show that PTEN-dependent phagocytosis is important for host protection during oral Lm infection. Overall, this study provides a comprehensive identification of macrophage factors involved in regulating Lm uptake and characterizes the function of one factor, PTEN, during Lm infection in vitro and in vivo. Importantly, these results demonstrate a role for opsonin-independent phagocytosis in Lm pathogenesis and suggest that macrophages play a primarily protective role during foodborne listeriosis.

Listeria monocytogenes utilizes glutathione and limited inorganic sulfur compounds as sources of essential cysteine.

Listeria monocytogenes (Lm) is a Gram-positive facultative intracellular pathogen that leads a biphasic lifecycle, transitioning its metabolism and selectively inducing virulence genes when it encounters mammalian hosts. Virulence gene expression is controlled by the master virulence regulator PrfA, which is allosterically activated by the host- and bacterially derived glutathione (GSH). The amino acid cysteine is the rate-limiting substrate for GSH synthesis in bacteria and is essential for bacterial growth. Unlike many bacteria, Lm is auxotrophic for cysteine and must import exogenous cysteine for growth and virulence. GSH is enriched in the host cytoplasm, and previous work suggests that Lm utilizes exogenous GSH for PrfA activation. Despite these observations, the import mechanism(s) for GSH remains elusive. Analysis of known GSH importers predicted a homologous importer in Lm comprised of the Ctp ABC transporter and the OppDF ATPases of the Opp oligopeptide importer. Here, we demonstrated that the Ctp complex is a high-affinity GSH/GSSG importer that is required for Lm growth at physiologically relevant concentrations. Furthermore, we demonstrated that OppDF is required for GSH/GSSG import in an Opp-independent manner. These data support a model where Ctp and OppDF form a unique complex for GSH/GSSG import that supports growth and pathogenesis. In addition, we show that Lm utilizes the inorganic sulfur sources thiosulfate and H2S for growth in a CysK-dependent manner in the absence of other cysteine sources. These findings suggest a pathoadaptive role for partial cysteine auxotrophy in Lm, where locally high GSH/GSSG or inorganic sulfur concentrations may signal arrival to distinct host niches.

The redox-responsive transcriptional regulator Rex represses fermentative metabolism and is required for Listeria monocytogenes pathogenesis.

The Gram-positive bacterium Listeria monocytogenes is the causative agent of the foodborne disease listeriosis, one of the deadliest bacterial infections known. In order to cause disease, L. monocytogenes must properly coordinate its metabolic and virulence programs in response to rapidly changing environments within the host. However, the mechanisms by which L. monocytogenes senses and adapts to the many stressors encountered as it transits through the gastrointestinal (GI) tract and disseminates to peripheral organs are not well understood. In this study, we investigated the role of the redox-responsive transcriptional regulator Rex in L. monocytogenes growth and pathogenesis. Rex is a conserved canonical transcriptional repressor that monitors the intracellular redox state of the cell by sensing the ratio of reduced and oxidized nicotinamide adenine dinucleotides (NADH and NAD+, respectively). Here, we demonstrated that L. monocytogenes Rex represses fermentative metabolism and is therefore required for optimal growth in the presence of oxygen. We also show that in vitro, Rex represses the production of virulence factors required for survival and invasion of the GI tract, as a strain lacking rex was more resistant to acidified bile and invaded host cells better than wild type. Consistent with these results, Rex was dispensable for colonizing the GI tract and disseminating to peripheral organs in an oral listeriosis model of infection. However, Rex-dependent regulation was required for colonizing the spleen and liver, and L. monocytogenes lacking the Rex repressor were nearly sterilized from the gallbladder. Taken together, these results demonstrated that Rex functions as a repressor of fermentative metabolism and suggests a role for Rex-dependent regulation in L. monocytogenes pathogenesis. Importantly, the gallbladder is the bacterial reservoir during listeriosis, and our data suggest redox sensing and Rex-dependent regulation are necessary for bacterial survival and replication in this organ.

The Transcriptional Regulator SpxA1 Influences the Morphology and Virulence of Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive facultative anaerobe and an excellent model pathogen for investigating regulatory changes that occur during infection of a mammalian host. SpxA1 is a widely conserved transcriptional regulator that induces expression of peroxide-detoxifying genes in L. monocytogenes and is thus required for aerobic growth. SpxA1 is also required for L. monocytogenes virulence, although the SpxA1-dependent genes important in this context remain to be identified. Here, we sought to investigate the role of SpxA1 in a tissue culture model of infection and made the surprising discovery that ΔspxA1 cells are dramatically elongated during growth in the host cytosol. Quantitative microscopy revealed that ΔspxA1 cells also form elongated filaments extracellularly during early exponential phase in rich medium. Scanning and transmission electron microscopy analysis found that the likely cause of this morphological phenotype is aberrantly placed division septa localized outside cell midpoints. Quantitative mass spectrometry of whole-cell lysates identified SpxA1-dependent changes in protein abundance, including a significant number of motility and flagellar proteins that were depleted in the ΔspxA1 mutant. Accordingly, we found that both the filamentation and the lack of motility contributed to decreased phagocytosis of ΔspxA1 cells by macrophages. Overall, we identify a novel role for SpxA1 in regulating cell elongation and motility, both of which impact L. monocytogenes virulence.

Listeria monocytogenes SpxA1 is a global regulator required to activate genes encoding catalase and heme biosynthesis enzymes for aerobic growth.

An imbalance of cellular oxidants and reductants causes redox stress, which must be rapidly detected to restore homeostasis. In bacteria, the Firmicutes encode conserved Spx-family transcriptional regulators that modulate transcription in response to redox stress. SpxA1 is an Spx-family orthologue in the intracellular pathogen Listeria monocytogenes that is essential for aerobic growth and pathogenesis. Here, we investigated the role of SpxA1 in growth and virulence by identifying genes regulated by SpxA1 in broth and during macrophage infection. We found SpxA1-activated genes encoding heme biosynthesis enzymes and catalase (kat) were required for L. monocytogenes aerobic growth in rich medium. An Spx-recognition motif previously defined in Bacillus subtilis was identified in the promoters of SpxA1-activated genes and proved necessary for the proper activation of two genes, indicating this regulation by SpxA1 is likely direct. Together, these findings elucidated the mechanism of spxA1 essentiality in vitro and demonstrated that SpxA1 is required for basal expression of scavenging enzymes to combat redox stress generated in the presence of oxygen.

Glutathione activates virulence gene expression of an intracellular pathogen.

Intracellular pathogens are responsible for much of the world-wide morbidity and mortality due to infectious diseases. To colonize their hosts successfully, pathogens must sense their environment and regulate virulence gene expression appropriately. Accordingly, on entry into mammalian cells, the facultative intracellular bacterial pathogen Listeria monocytogenes remodels its transcriptional program by activating the master virulence regulator PrfA. Here we show that bacterial and host-derived glutathione are required to activate PrfA. In this study a genetic selection led to the identification of a bacterial mutant in glutathione synthase that exhibited reduced virulence gene expression and was attenuated 150-fold in mice. Genome sequencing of suppressor mutants that arose spontaneously in vivo revealed a single nucleotide change in prfA that locks the protein in the active conformation (PrfA*) and completely bypassed the requirement for glutathione during infection. Biochemical and genetic studies support a model in which glutathione-dependent PrfA activation is mediated by allosteric binding of glutathione to PrfA. Whereas glutathione and other low-molecular-weight thiols have important roles in redox homeostasis in all forms of life, here we demonstrate that glutathione represents a critical signalling molecule that activates the virulence of an intracellular pathogen.

YjbH Requires Its Thioredoxin Active Motif for the Nitrosative Stress Response, Cell-to-Cell Spread, and Protein-Protein Interactions in Listeria monocytogenes.

Listeria monocytogenes is a model facultative intracellular pathogen. Tight regulation of virulence proteins is essential for a successful infection, and the gene encoding the annotated thioredoxin YjbH was identified in two forward genetic screens as required for virulence factor production. Accordingly, an L. monocytogenes strain lacking yjbH is attenuated in a murine model of infection. However, the function of YjbH in L. monocytogenes has not been investigated. Here, we provide evidence that L. monocytogenes YjbH is involved in the nitrosative stress response, likely through its interaction with the redox-responsive transcriptional regulator SpxA1. YjbH physically interacted with SpxA1, and our data support a model in which YjbH is a protease adaptor that regulates SpxA1 protein abundance. Whole-cell proteomics identified eight additional proteins whose abundance was altered by YjbH, and we demonstrated that YjbH physically interacted with each in bacterial two-hybrid assays. Thioredoxin proteins canonically require active motif cysteines for function, but thioredoxin activity has not been tested for L. monocytogenes YjbH. We demonstrated that cysteine residues of the YjbH thioredoxin domain active motif are essential for L. monocytogenes sensitivity to nitrosative stress, cell-to-cell spread in a tissue culture model of infection, and several protein-protein interactions. Together, these results demonstrated that the function of YjbH in L. monocytogenes requires its thioredoxin active motif and that YjbH has a role in the posttranslational regulation of several proteins, including SpxA1.IMPORTANCE The annotated thioredoxin YjbH in Listeria monocytogenes has been implicated in virulence, but its function in the cell is unknown. In other bacterial species, YjbH is a protease adaptor that mediates degradation of the transcriptional regulator Spx. Here, we investigated the function of L. monocytogenes YjbH and demonstrated its role in the nitrosative stress response and posttranslational regulation of several proteins with which YjbH physically interacts, including SpxA1. Furthermore, we demonstrated that the cysteine residues of the YjbH thioredoxin active motif are required for the nitrosative stress response, cell-to-cell spread, and some protein-protein interactions. YjbH is widely conserved among Firmicutes, and this work reveals its unique requirement of the thioredoxin-active motif in L. monocytogenes.

Reduce, Induce, Thrive: Bacterial Redox Sensing during Pathogenesis.

The abundance of oxidants and reductants must be balanced for an organism to thrive. Bacteria have evolved methods to prevent redox imbalances and to mitigate their deleterious consequences through the expression of detoxification enzymes, antioxidants, and systems to repair or degrade damaged proteins and DNA. Regulating these processes in response to redox changes requires sophisticated surveillance strategies ranging from metal chelation to direct sensing of toxic reactive oxygen species. In the case of bacterial pathogens, stress that threatens to disrupt redox homeostasis can derive from endogenous sources (produced by the bacteria) or exogenous sources (produced by the host). This minireview summarizes the sources of redox stress encountered during infection, the mechanisms by which bacterial pathogens diminish the damaging effects of redox stress, and the clever ways some organisms have evolved to thrive in the face of redox challenges during infection.

An In Vivo Selection Identifies Listeria monocytogenes Genes Required to Sense the Intracellular Environment and Activate Virulence Factor Expression.

Listeria monocytogenes is an environmental saprophyte and facultative intracellular bacterial pathogen with a well-defined life-cycle that involves escape from a phagosome, rapid cytosolic growth, and ActA-dependent cell-to-cell spread, all of which are dependent on the master transcriptional regulator PrfA. The environmental cues that lead to temporal and spatial control of L. monocytogenes virulence gene expression are poorly understood. In this study, we took advantage of the robust up-regulation of ActA that occurs intracellularly and expressed Cre recombinase from the actA promoter and 5' untranslated region in a strain in which loxP sites flanked essential genes, so that activation of actA led to bacterial death. Upon screening for transposon mutants that survived intracellularly, six genes were identified as necessary for ActA expression. Strikingly, most of the genes, including gshF, spxA1, yjbH, and ohrA, are predicted to play important roles in bacterial redox regulation. The mutants identified in the genetic selection fell into three broad categories: (1) those that failed to reach the cytosolic compartment; (2) mutants that entered the cytosol, but failed to activate the master virulence regulator PrfA; and (3) mutants that entered the cytosol and activated transcription of actA, but failed to synthesize it. The identification of mutants defective in vacuolar escape suggests that up-regulation of ActA occurs in the host cytosol and not the vacuole. Moreover, these results provide evidence for two non-redundant cytosolic cues; the first results in allosteric activation of PrfA via increased glutathione levels and transcriptional activation of actA while the second results in translational activation of actA and requires yjbH. Although the precise host cues have not yet been identified, we suggest that intracellular redox stress occurs as a consequence of both host and pathogen remodeling their metabolism upon infection.

A Redox-Responsive Transcription Factor Is Critical for Pathogenesis and Aerobic Growth of Listeria monocytogenes.

Bacterial pathogens have evolved sophisticated mechanisms to sense and adapt to redox stress in nature and within the host. However, deciphering the redox environment encountered by intracellular pathogens in the mammalian cytosol is challenging, and that environment remains poorly understood. In this study, we assessed the contributions of the two redox-responsive, Spx-family transcriptional regulators to the virulence of Listeria monocytogenes, a Gram-positive facultative intracellular pathogen. Spx-family proteins are highly conserved in Firmicutes, and the L. monocytogenes genome contains two paralogues, spxA1 and spxA2 Here, we demonstrate that spxA1, but not spxA2, is required for the oxidative stress response and pathogenesis. SpxA1 function appeared to be conserved with the Bacillus subtilis homologue, and resistance to oxidative stress required the canonical CXXC redox-sensing motif. Remarkably, spxA1 was essential for aerobic growth, demonstrating that L. monocytogenes SpxA1 likely regulates a distinct set of genes. Although the ΔspxA1 mutant did not grow in the presence of oxygen in the laboratory, it was able to replicate in macrophages and colonize the spleens, but not the livers, of infected mice. These data suggest that the redox state of bacteria during infection differs significantly from that of bacteria growing in vitro Further, the host cell cytosol may resemble an anaerobic environment, with tissue-specific variations in redox stress and oxygen concentration.

Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation.

Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type in vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection model in vivo This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

Selective pharmacologic inhibition of a PASTA kinase increases Listeria monocytogenes susceptibility to ß-lactam antibiotics.

While ß-lactam antibiotics are a critical part of the antimicrobial arsenal, they are frequently compromised by various resistance mechanisms, including changes in penicillin binding proteins of the bacterial cell wall. Genetic deletion of the penicillin binding protein and serine/threonine kinase-associated protein (PASTA) kinase in methicillin-resistant Staphylococcus aureus (MRSA) has been shown to restore ß-lactam susceptibility. However, the mechanism remains unclear, and whether pharmacologic inhibition would have the same effect is unknown. In this study, we found that deletion or pharmacologic inhibition of the PASTA kinase in Listeria monocytogenes by the nonselective kinase inhibitor staurosporine results in enhanced susceptibility to both aminopenicillin and cephalosporin antibiotics. Resistance to vancomycin, another class of cell wall synthesis inhibitors, or antibiotics that inhibit protein synthesis was unaffected by staurosporine treatment. Phosphorylation assays with purified kinases revealed that staurosporine selectively inhibited the PASTA kinase of L. monocytogenes (PrkA). Importantly, staurosporine did not inhibit a L. monocytogenes kinase without a PASTA domain (Lmo0618) or the PASTA kinase from MRSA (Stk1). Finally, inhibition of PrkA with a more selective kinase inhibitor, AZD5438, similarly led to sensitization of L. monocytogenes to ß-lactam antibiotics. Overall, these results suggest that pharmacologic targeting of PASTA kinases can increase the efficacy of ß-lactam antibiotics.

Analysis of genetic requirements and nutrient availability for Staphylococcus aureus growth in cystic fibrosis sputum.

Staphylococcus aureus is one of the most common pathogens isolated from the lungs of people with cystic fibrosis (CF), but little is known about its ability to colonize this niche. We performed a Tn-seq screen to identify genes necessary for S. aureus growth in media prepared from ex vivo CF sputum. We identified 19 genes that were required for growth in all sputum media tested and dozens more that were required for growth in at least one sputum medium. Depleted mutants of interest included insertions in many genes important for surviving metal starvation as well as the primary regulator of cysteine metabolism cymR. To investigate the mechanisms by which these genes contribute to S. aureus growth in sputum, we quantified low-molecular-weight thiols, nutrient transition metals, and the host metal-sequestration protein calprotectin in sputum from 11 individuals with CF. In all samples, the abundance of calprotectin exceeded nutrient metal concentration, explaining the S. aureus requirement for metal-starvation genes. Further, all samples contain potentially toxic quantities of cysteine and sufficient glutathione to satisfy the organic sulfur requirements of S. aureus. Deletion of the cysteine importer genes tcyA and tcyP in the ∆cymR background restored growth to wild-type levels in CF sputum, suggesting that the mechanism by which cymR is required for growth in sputum is to prevent uncontrolled import of cysteine or cystine from this environment. Overall, this work demonstrates that calprotectin and cysteine limit S. aureus growth in CF sputum.

Regulation of host hemoglobin binding by the Staphylococcus aureus Clp proteolytic system.

Protein turnover is a key process for bacterial survival mediated by intracellular proteases. Proteolytic degradation reduces the levels of unfolded and misfolded peptides that accumulate in the cell during stress conditions. Three intracellular proteases, ClpP, HslV, and FtsH, have been identified in the Gram-positive bacterium Staphylococcus aureus, a pathogen responsible for significant morbidity and mortality worldwide. Consistent with their crucial role in protein turnover, ClpP, HslV, and FtsH affect a number of cellular processes, including metabolism, stress responses, and virulence. The ClpP protease is believed to be the principal degradation machinery in S. aureus. This study sought to identify the effect of the Clp protease on the iron-regulated surface determinant (Isd) system, which extracts heme-iron from host hemoglobin during infection and is critical to S. aureus pathogenesis. Inactivation of components of the Clp protease alters abundance of several Isd proteins, including the hemoglobin receptor IsdB. Furthermore, the observed changes in IsdB abundance are the result of transcriptional regulation, since transcription of isdB is decreased by clpP or clpX inactivation. In contrast, inactivation of clpC enhances isdB transcription and protein abundance. Loss of clpP or clpX impairs host hemoglobin binding and utilization and results in severe virulence defects in a systemic mouse model of infection. These findings suggest that the Clp proteolytic system is important for regulating nutrient iron acquisition in S. aureus. The Clp protease and Isd complex are widely conserved in bacteria; therefore, these data reveal a novel Clp-dependent regulation pathway that may be present in other bacterial pathogens.

Small molecule inhibitors of Staphylococcus aureus RnpA alter cellular mRNA turnover, exhibit antimicrobial activity, and attenuate pathogenesis.

Methicillin-resistant Staphylococcus aureus is estimated to cause more U.S. deaths annually than HIV/AIDS. The emergence of hypervirulent and multidrug-resistant strains has further amplified public health concern and accentuated the need for new classes of antibiotics. RNA degradation is a required cellular process that could be exploited for novel antimicrobial drug development. However, such discovery efforts have been hindered because components of the Gram-positive RNA turnover machinery are incompletely defined. In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro. Exploiting this activity, high through-put and secondary screening assays identified a small molecule inhibitor of RnpA-mediated in vitro RNA degradation. This agent was shown to limit cellular mRNA degradation and exhibited antimicrobial activity against predominant methicillin-resistant S. aureus (MRSA) lineages circulating throughout the U.S., vancomycin intermediate susceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) and other Gram-positive bacterial pathogens with high RnpA amino acid conservation. We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus. Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy.

Listeria monocytogenes folate metabolism is required to generate N-formylmethionine during infection.

Folic acid and its derivatives are required for the synthesis of purines, pyrimidines, and some amino acids. Antifolate antibiotics that target the folic acid metabolism pathway are commonly used for the treatment of listeriosis caused by the intracellular pathogen Listeria monocytogenes (Lm). In recent work in mBio, Feng et al. sought to understand the role of folic acid metabolism in Lm virulence (Y. Feng, S. Chang, D. A. Portnoy, 2023, mBio https://doi.org/10.1128/mbio.01074-23). The authors discovered that N-formylmethionine, an amino acid utilized by bacteria to initiate protein synthesis, is crucial for Lm intracellular growth and pathogenesis. Surprisingly, purines and thymidine were found to be dispensable for Lm infection. Together these results demonstrate that while Lm can obtain many essential nutrients from the host cytosol, including purines and most amino acids, it requires N-formylmethionine biosynthesis to properly regulate translation initiation during infection.

Listeria monocytogenes utilizes glutathione and limited inorganic sulfur compounds as a source of essential L-cysteine.

Listeria monocytogenes ( Lm ) is a Gram-positive facultative intracellular pathogen that leads a biphasic lifecycle, transitioning its metabolism and selectively inducing virulence genes when it encounters mammalian hosts. Virulence gene expression is controlled by the master virulence regulator PrfA, which is allosterically activated by host- and bacterially-derived glutathione (GSH). The amino acid L-cysteine is the rate-limiting substrate for GSH synthesis in bacteria and is essential for bacterial growth. Unlike many bacteria, Lm is auxotrophic for L-cysteine and must import exogenous cysteine for growth and virulence. GSH is enriched in the host cytoplasm, and previous work suggests that Lm utilizes exogenous GSH for PrfA activation. Despite these observations, the import mechanism(s) for GSH remains elusive. Analysis of known GSH importers predicted a homologous importer in Lm comprised of the Ctp ABC transporter and the OppDF ATPases of the Opp oligopeptide importer. Here, we demonstrated that the Ctp complex is a high-affinity GSH/GSSG importer that is required for Lm growth at physiologically relevant concentrations. Further, we demonstrated that OppDF are required for GSH/GSSG import in an Opp-independent manner. These data support a model where Ctp and OppDF form a unique complex for GSH/GSSG import that supports growth and pathogenesis. Additionally, we show that Lm utilizes the inorganic sulfur sources thiosulfate and H 2 S for growth in a CysK-dependent manner in the absence of other L-cysteine sources. These findings suggest a pathoadaptive role for partial cysteine auxotrophy in Lm , where locally high GSH/GSSG or inorganic sulfur concentrations may signal arrival to distinct host niches.

Genome-wide protein-DNA interaction site mapping in bacteria using a double-stranded DNA-specific cytosine deaminase.

DNA-protein interactions are central to fundamental cellular processes, yet widely implemented technologies for measuring these interactions on a genome scale in bacteria are laborious and capture only a snapshot of binding events. We devised a facile method for mapping DNA-protein interaction sites in vivo using the double-stranded DNA-specific cytosine deaminase toxin DddA. In 3D-seq (DddA-sequencing), strains containing DddA fused to a DNA-binding protein of interest accumulate characteristic mutations in DNA sequence adjacent to sites occupied by the DNA-bound fusion protein. High-depth sequencing enables detection of sites of increased mutation frequency in these strains, yielding genome-wide maps of DNA-protein interaction sites. We validated 3D-seq for four transcription regulators in two bacterial species, Pseudomonas aeruginosa and Escherichia coli. We show that 3D-seq offers ease of implementation, the ability to record binding event signatures over time and the capacity for single-cell resolution.

The flexible loop of Staphylococcus aureus IsdG is required for its degradation in the absence of heme.

Degradation of specific native proteins allows bacteria to rapidly adapt to changing environments when the activity of those proteins is no longer required. Although these processes are vital to bacterial survival, relatively little is known regarding how bacterial proteins are recognized and targeted for degradation. Staphylococcus aureus is an important human pathogen that requires iron for growth and pathogenesis. In the vertebrate host, S. aureus fulfills its iron requirement by obtaining heme iron from host hemoproteins via IsdG- and IsdI-mediated heme degradation. IsdG and IsdI are structurally and mechanistically analogous but are differentially regulated by iron and heme availability. Specifically, IsdG is targeted for degradation in the absence of heme. Therefore, we utilized the differential regulation of IsdG and IsdI to investigate the mechanism of regulated proteolysis. In contrast to canonical protease recognition sequences, we show that IsdG is targeted for degradation by internally coded sequences. Specifically, a flexible loop near the heme-binding pocket is required for IsdG degradation in the absence of heme.

The IsdG-family of haem oxygenases degrades haem to a novel chromophore.

Enzymatic haem catabolism by haem oxygenases is conserved from bacteria to humans and proceeds through a common mechanism leading to the formation of iron, carbon monoxide and biliverdin. The first members of a novel class of haem oxygenases were recently identified in Staphylococcus aureus (IsdG and IsdI) and were termed the IsdG-family of haem oxygenases. Enzymes of the IsdG-family form tertiary structures distinct from those of the canonical haem oxygenase family, suggesting that IsdG-family members degrade haem via a unique reaction mechanism. Herein we report that the IsdG-family of haem oxygenases degrade haem to the oxo-bilirubin chromophore staphylobilin. We also present the crystal structure of haem-bound IsdI in which haem ruffling and constrained binding of oxygen is consistent with cleavage of the porphyrin ring at the beta- or delta-meso carbons. Combined, these data establish that the IsdG-family of haem oxygenases degrades haem to a novel chromophore distinct from biliverdin.