Lipidomics of homeoviscous adaptation to low temperatures in Staphylococcus aureus utilizing exogenous straight-chain unsaturated fatty acids.

It is well established that Staphylococcus aureus can incorporate exogenous straight-chain unsaturated fatty acids (SCUFAs) into membrane phospho- and glyco-lipids from various sources in supplemented culture media and when growing in vivo during infection. Given the enhancement of membrane fluidity when oleic acid (C18:1Δ9) is incorporated into lipids, we were prompted to examine the effect of medium supplementation with C18:1Δ9 on growth at low temperatures. C18:1Δ9 supported the growth of a cold-sensitive, branched-chain fatty acid (BCFA)-deficient mutant at 12°C. Interestingly, we found similar results in the BCFA-sufficient parental strain, supported by the fact that the incorporation of C18:1Δ9 into the membrane increased membrane fluidity in both strains. We show that the incorporation of C18:1Δ9 and its elongation product C20:1Δ11 into membrane lipids was required for growth stimulation and relied on a functional FakAB incorporation system. Lipidomics analysis of the phosphatidylglycerol and diglycosyldiacylglycerol lipid classes revealed major impacts of C18:1Δ9 and temperature on lipid species. Growth at 12°C in the presence of C18:1Δ9 also led to increased production of the carotenoid pigment staphyloxanthin. The enhancement of growth by C18:1Δ9 is an example of homeoviscous adaptation to low temperatures utilizing an exogenous fatty acid. This may be significant in the growth of S. aureus at low temperatures in foods that commonly contain C18:1Δ9 and other SCUFAs in various forms. IMPORTANCE: We show that Staphylococcus aureus can use its known ability to incorporate exogenous fatty acids to enhance its growth at low temperatures. Individual species of phosphatidylglycerols and diglycosyldiacylglycerols bearing one or two degrees of unsaturation derived from the incorporation of C18:1Δ9 at 12°C are described for the first time. In addition, enhanced production of the carotenoid staphyloxanthin occurs at low temperatures. The studies describe a biochemical reality underlying membrane biophysics. This is an example of homeoviscous adaptation to low temperatures utilizing exogenous fatty acids over the regulation of the biosynthesis of endogenous fatty acids. The studies have likely relevance to food safety in that unsaturated fatty acids may enhance the growth of S. aureus in the food environment.

Polyphosphate: A Conserved Modifier of Amyloidogenic Processes.

Polyphosphate (polyP), a several billion-year-old biopolymer, is produced in every cell, tissue, and organism studied. Structurally extremely simple, polyP consists of long chains of covalently linked inorganic phosphate groups. We report here the surprising discovery that polyP shows a remarkable efficacy in accelerating amyloid fibril formation. We found that polyP serves as an effective nucleation source for various different amyloid proteins, ranging from bacterial CsgA to human α-synuclein, Aß1-40/42, and Tau. polyP-associated α-synuclein fibrils show distinct differences in seeding behavior, morphology, and fibril stability compared with fibrils formed in the absence of polyP. In vivo, the amyloid-stimulating and fibril-stabilizing effects of polyP have wide-reaching consequences, increasing the rate of biofilm formation in pathogenic bacteria and mitigating amyloid toxicity in differentiated neuroblastoma cells and C. elegans strains that serve as models for human folding diseases. These results suggest that we have discovered a conserved cytoprotective modifier of amyloidogenic processes.

Expression of RcrB confers resistance to hypochlorous acid in uropathogenic Escherichia coli.

To eradicate bacterial pathogens, neutrophils are recruited to the sites of infection, where they engulf and kill microbes through the production of reactive oxygen and chlorine species (ROS/RCS). The most prominent RCS is the antimicrobial oxidant hypochlorous acid (HOCl), which rapidly reacts with various amino acid side chains, including those containing sulfur and primary/tertiary amines, causing significant macromolecular damage. Pathogens like uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, have developed sophisticated defense systems to protect themselves from HOCl. We recently identified the RcrR regulon as a novel HOCl defense strategy in UPEC. Expression of the rcrARB operon is controlled by the HOCl-sensing transcriptional repressor RcrR, which is oxidatively inactivated by HOCl resulting in the expression of its target genes, including rcrB. The rcrB gene encodes a hypothetical membrane protein, deletion of which substantially increases UPEC's susceptibility to HOCl. However, the mechanism behind protection by RcrB is unclear. In this study, we investigated whether (i) its mode of action requires additional help, (ii) rcrARB expression is induced by physiologically relevant oxidants other than HOCl, and (iii) expression of this defense system is limited to specific media and/or cultivation conditions. We provide evidence that RcrB expression is sufficient to protect E. coli from HOCl. Furthermore, RcrB expression is induced by and protects from several RCS but not from ROS. RcrB plays a protective role for RCS-stressed planktonic cells under various growth and cultivation conditions but appears to be irrelevant for UPEC's biofilm formation. IMPORTANCE Bacterial infections pose an increasing threat to human health, exacerbating the demand for alternative treatments. Uropathogenic Escherichia coli (UPEC), the most common etiological agent of urinary tract infections (UTIs), are confronted by neutrophilic attacks in the bladder, and must therefore be equipped with powerful defense systems to fend off the toxic effects of reactive chlorine species. How UPEC deal with the negative consequences of the oxidative burst in the neutrophil phagosome remains unclear. Our study sheds light on the requirements for the expression and protective effects of RcrB, which we recently identified as UPEC's most potent defense system toward hypochlorous acid (HOCl) stress and phagocytosis. Thus, this novel HOCl stress defense system could potentially serve as an attractive drug target to increase the body's own capacity to fight UTIs.

Bacterial Defense Systems against the Neutrophilic Oxidant Hypochlorous Acid.

Neutrophils kill invading microbes and therefore represent the first line of defense of the innate immune response. Activated neutrophils assemble NADPH oxidase to convert substantial amounts of molecular oxygen into superoxide, which, after dismutation into peroxide, serves as the substrate for the generation of the potent antimicrobial hypochlorous acid (HOCl) in the phagosomal space. In this minireview, we explore the most recent insights into physiological consequences of HOCl stress. Not surprisingly, Gram-negative bacteria have evolved diverse posttranslational defense mechanisms to protect their proteins, the main targets of HOCl, from HOCl-mediated damage. We discuss the idea that oxidation of conserved cysteine residues and partial unfolding of its structure convert the heat shock protein Hsp33 into a highly active chaperone holdase that binds unfolded proteins and prevents their aggregation. We examine two novel members of the Escherichia coli chaperone holdase family, RidA and CnoX, whose thiol-independent activation mechanism differs from that of Hsp33 and requires N-chlorination of positively charged amino acids during HOCl exposure. Furthermore, we summarize the latest findings with respect to another bacterial defense strategy employed in response to HOCl stress, which involves the accumulation of the universally conserved biopolymer inorganic polyphosphate. We then discuss sophisticated adaptive strategies that bacteria have developed to enhance their survival during HOCl stress. Understanding bacterial defense and survival strategies against one of the most powerful neutrophilic oxidants may provide novel insights into treatment options that potentially compromise the ability of pathogens to resist HOCl stress and therefore may increase the efficacy of the innate immune response.

Pseudomonas aeruginosa defense systems against microbicidal oxidants.

The most abundant oxidants controlling bacterial colonization on mucosal barrier epithelia are hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN). All three oxidants are highly antimicrobial but little is known about their relative efficacies, their respective cellular targets, or what specific responses they elicit in bacteria. To address these important questions, we directly tested the individual oxidants on the virulent Pseudomonas aeruginosa strain PA14. We discovered that HOCl and HOBr work almost interchangeably, impacting non-growing bacterial cultures more significantly than actively growing bacteria, and eliciting similar stress responses, including the heat shock response. HOSCN treatment is distinctly different, affecting primarily actively growing PA14 and evoking stress responses suggestive of membrane damage. What all three oxidants have in common, however, is their ability to cause substantial protein aggregation. This effect became particularly obvious in strains lacking polyphosphate, a newly recognized chemical chaperone. Treatment of PA14 with the FDA-approved anti-inflammatory drug mesalamine, which has recently been shown to attenuate polyP production in a wide range of bacteria, effectively decreased the resistance of PA14 toward all three oxidants, suggesting that we have discovered a novel, targetable defense system in P. aeruginosa.

HdeB functions as an acid-protective chaperone in bacteria.

Enteric bacteria such as Escherichia coli utilize various acid response systems to counteract the acidic environment of the mammalian stomach. To protect their periplasmic proteome against rapid acid-mediated damage, bacteria contain the acid-activated periplasmic chaperones HdeA and HdeB. Activation of HdeA at pH 2 was shown to correlate with its acid-induced dissociation into partially unfolded monomers. In contrast, HdeB, which has high structural similarities to HdeA, shows negligible chaperone activity at pH 2 and only modest chaperone activity at pH 3. These results raised intriguing questions concerning the physiological role of HdeB in bacteria, its activation mechanism, and the structural requirements for its function as a molecular chaperone. In this study, we conducted structural and biochemical studies that revealed that HdeB indeed works as an effective molecular chaperone. However, in contrast to HdeA, whose chaperone function is optimal at pH 2, the chaperone function of HdeB is optimal at pH 4, at which HdeB is still fully dimeric and largely folded. NMR, analytical ultracentrifugation, and fluorescence studies suggest that the highly dynamic nature of HdeB at pH 4 alleviates the need for monomerization and partial unfolding. Once activated, HdeB binds various unfolding client proteins, prevents their aggregation, and supports their refolding upon subsequent neutralization. Overexpression of HdeA promotes bacterial survival at pH 2 and 3, whereas overexpression of HdeB positively affects bacterial growth at pH 4. These studies demonstrate how two structurally homologous proteins with seemingly identical in vivo functions have evolved to provide bacteria with the means for surviving a range of acidic protein-unfolding conditions.

The sulfur carrier protein TusA has a pleiotropic role in Escherichia coli that also affects molybdenum cofactor biosynthesis.

The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes.

Lipidomics of homeoviscous adaptation to low temperatures in Staphylococcus aureus utilizing exogenous straight-chain unsaturated fatty acids over biosynthesized endogenous branched-chain fatty acids.

It is well established that Staphylococcus aureus can incorporate exogenous straight-chain unsaturated fatty acids (SCUFAs) into membrane phospho- and glyco-lipids from various sources in supplemented culture media, and when growing in vivo in an infection. Given the enhancement of membrane fluidity when oleic acid (C18:1Δ9) is incorporated into lipids, we were prompted to examine the effect of medium supplementation with C18:1Δ9 on growth at low temperatures. C18:1Δ9 supported the growth of a cold-sensitive, branched-chain fatty acid (BCFA)-deficient mutant at 12°C. Interestingly, we found similar results in the BCFA-sufficient parental strain. We show that incorporation of C18:1Δ9 and its elongation product C20:1Δ9 into membrane lipids was required for growth stimulation and relied on a functional FakAB incorporation system. Lipidomics analysis of the phosphatidylglycerol (PG) and diglycosyldiacylglycerol (DGDG) lipid classes revealed major impacts of C18:1Δ9 and temperature on lipid species. Growth at 12°C in the presence of C18:1Δ9 also led to increased production of the carotenoid pigment staphyloxanthin; however, this was not an obligatory requirement for cold adaptation. Enhancement of growth by C18:1Δ9 is an example of homeoviscous adaptation to low temperatures utilizing an exogenous fatty acid. This may be significant in the growth of S. aureus at low temperatures in foods that commonly contain C18:1Δ9 and other SCUFAs in various forms.

A Year at the Forefront of Bacterial Defense Systems Against Neutrophilic Oxidants.

One challenge for invading pathogens represents the exposure to highly microbicidal hypohalous acids (HOX), such as hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Generated at high concentrations by innate immune cells during phagocytosis, HOX kills the engulfed microbes through extensive macromolecular damage. However, microorganisms have evolved strategies to detoxify the oxidants and/or alleviate HOX-mediated damage, which improves their survival during HOX exposure. Many of these defense systems are bacteria-specific and therefore considered potential drug targets. Our minireview highlights recent (July 2021 to November 2022) advances in the field of microbial HOX defense systems and how these systems are regulated. We report recent progress on redox-sensing transcriptional regulators, two-component systems, and σ/anti-σ factors and review how oxidative modifications in these regulatory proteins affect the expression of their target genes. Moreover, we discuss novel studies that describe how HOCl affects the activity of redox-regulated enzymes and highlight mechanisms that bacteria employ to reduce HOSCN.

Pseudomonas aeruginosa kills Staphylococcus aureus in a polyphosphate-dependent manner.

Due to their frequent coexistence in many polymicrobial infections, including in patients with burn or chronic wounds or cystic fibrosis, recent studies have started to investigate the mechanistic details of the interaction between the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus. P. aeruginosa rapidly outcompetes S. aureus under in vitro co-cultivation conditions, which is mediated by several of P. aeruginosa's virulence factors. Here, we report that polyphosphate (polyP), an efficient stress defense system and virulence factor in P. aeruginosa, plays a role for the pathogen's ability to inhibit and kill S. aureus in a contact-independent manner. We show that P. aeruginosa cells characterized by low polyP level are less detrimental to S. aureus growth and survival while the gram-positive pathogen is significantly more compromised by the presence of P. aeruginosa cells that produce high level of polyP. We show that the polyP-dependent phenotype could be a direct effect by the biopolymer, as polyP is present in the spent media and causes significant damage to the S. aureus cell envelope. However, more likely is that polyP's effects are indirect through the regulation of one of P. aeruginosa's virulence factors, pyocyanin. We show that pyocyanin production in P. aeruginosa occurs polyP-dependent and harms S. aureus through membrane damage and the generation of reactive oxygen species, resulting in increased expression of antioxidant enzymes. In summary, our study adds a new component to the list of biomolecules that the gram-negative pathogen P. aeruginosa generates to compete with S. aureus for resources.

A novel ruthenium-silver based antimicrobial potentiates aminoglycoside activity against Pseudomonas aeruginosa.

The rapid dissemination of antibiotic resistance combined with the decline in the discovery of novel antibiotics represents a major challenge for infectious disease control that can only be mitigated by investments in novel treatment strategies. Alternative antimicrobials, including silver, have regained interest due to their diverse mechanisms of inhibiting microbial growth. One such example is AGXX, a broad-spectrum antimicrobial that produces highly cytotoxic reactive oxygen species (ROS) to inflict extensive macromolecular damage. Due to the connections identified between ROS production and antibiotic lethality, we hypothesized that AGXX could potentially increase the activity of conventional antibiotics. Using the gram-negative pathogen Pseudomonas aeruginosa, we screened possible synergistic effects of AGXX on several antibiotic classes. We found that the combination of AGXX and aminoglycosides tested at sublethal concentrations led to a rapid exponential decrease in bacterial survival and restored the sensitivity of a kanamycin-resistant strain. ROS production contributes significantly to the bactericidal effects of AGXX/aminoglycoside treatments, which is dependent on oxygen availability and can be reduced by the addition of ROS scavengers. Additionally, P. aeruginosa strains deficient in ROS detoxifying/repair genes were more susceptible to AGXX/aminoglycoside treatment. We further demonstrate that this synergistic interaction was associated with a significant increase in outer and inner membrane permeability, resulting in increased antibiotic influx. Our study also revealed that AGXX/aminoglycoside-mediated killing requires an active proton motive force across the bacterial membrane. Overall, our findings provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials. IMPORTANCE The emergence of drug-resistant bacteria coupled with the decline in antibiotic development highlights the need for novel alternatives. Thus, new strategies aimed at repurposing conventional antibiotics have gained significant interest. The necessity of these interventions is evident especially in gram-negative pathogens as they are particularly difficult to treat due to their outer membrane. This study highlights the effectiveness of the antimicrobial AGXX in potentiating aminoglycoside activities against P. aeruginosa. The combination of AGXX and aminoglycosides not only reduces bacterial survival rapidly but also significantly re-sensitizes aminoglycoside-resistant P. aeruginosa strains. In combination with gentamicin, AGXX induces increased endogenous oxidative stress, membrane damage, and iron-sulfur cluster disruption. These findings emphasize AGXX's potential as a route of antibiotic adjuvant development and shed light on potential targets to enhance aminoglycoside activity.

The Novel Silver-Containing Antimicrobial Potentiates Aminoglycoside Activity Against Pseudomonas aeruginosa.

The rapid dissemination of antibiotic resistance combined with the decline in the discovery of novel antibiotics represents a major challenge for infectious disease control that can only be mitigated by investments into novel treatment strategies. Alternative antimicrobials, including silver, have regained interest due to their diverse mechanisms of inhibiting microbial growth. One such example is AGXX®, a broad-spectrum silver containing antimicrobial that produces highly cytotoxic reactive oxygen species (ROS) to inflict extensive macromolecular damage. Due to connections identified between ROS production and antibiotic lethality, we hypothesized that AGXX® could potentially increase the activity of conventional antibiotics. Using the gram-negative pathogen Pseudomonas aeruginosa, we screened possible synergistic effects of AGXX® on several antibiotic classes. We found that the combination of AGXX® and aminoglycosides tested at sublethal concentrations led to a rapid exponential decrease in bacterial survival and restored sensitivity of a kanamycin-resistant strain. ROS production contributes significantly to the bactericidal effects of AGXX®/aminoglycoside treatments, which is dependent on oxygen availability and can be reduced by the addition of ROS scavengers. Additionally, P. aeruginosa strains deficient in ROS detoxifying/repair genes were more susceptible to AGXX®/aminoglycoside treatment. We further demonstrate that this synergistic interaction was associated with significant increase in outer and inner membrane permeability, resulting in increased antibiotic influx. Our study also revealed that AGXX®/aminoglycoside-mediated killing requires an active proton motive force across the bacterial membrane. Overall, our findings provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials.

A Social Media Intervention for Promoting Oral Health Behaviors in Adolescents: A Non-Randomized Pilot Clinical Trial.

Poor oral hygiene and excessive consumption of soda are among the main drivers of systemic health issues in adolescents in the United States. This non-randomized pilot clinical trial focused on the effects of a health text message system and smartphone-based intervention on adolescent tooth-brushing behavior and dietary choices, with a convenience sample of 94 participants aged 12 to 14 years old. A group of 75 participants agreed to use a tooth-brushing app and received a health text message; the other group of 15 agreed to use the tooth-brushing app, but did not receive a health text message. Saliva specimens were collected directly before and at the end of each experiment; changes in the salivary presence of cariogenic bacteria over the duration of the study were evaluated and compared with the demographics and behavioral variables. Within the text message group, 5% of participants increased the frequency of daily tooth brushing. Within the non-intervention group, 29% of participants increased the frequency of their daily tooth brushing. There were reductions in the total salivary bacteria and total streptococci in both groups (p < 0.001), but no change in the presence of cariogenic Mutans streptococci. Raising adolescents' consciousness of oral health behavior resulted in marginal to moderate improvements to oral hygiene and dietary choices, as well as reductions in total salivary bacteria.

Expression of RcrB confers resistance to hypochlorous acid in uropathogenic Escherichia coli.

To eradicate bacterial pathogens, neutrophils are recruited to the sites of infection, where they engulf and kill microbes through the production of reactive oxygen and chlorine species (ROS/RCS). The most prominent RCS is antimicrobial oxidant hypochlorous acid (HOCl), which rapidly reacts with various amino acids side chains, including those containing sulfur and primary/tertiary amines, causing significant macromolecular damage. Pathogens like uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections (UTIs), have developed sophisticated defense systems to protect themselves from HOCl. We recently identified the RcrR regulon as a novel HOCl defense strategy in UPEC. The regulon is controlled by the HOCl-sensing transcriptional repressor RcrR, which is oxidatively inactivated by HOCl resulting in the expression of its target genes, including rcrB . rcrB encodes the putative membrane protein RcrB, deletion of which substantially increases UPEC's susceptibility to HOCl. However, many questions regarding RcrB's role remain open including whether (i) the protein's mode of action requires additional help, (ii) rcrARB expression is induced by physiologically relevant oxidants other than HOCl, and (iii) expression of this defense system is limited to specific media and/or cultivation conditions. Here, we provide evidence that RcrB expression is sufficient to E. coli 's protection from HOCl and induced by and protects from several RCS but not from ROS. RcrB plays a protective role for RCS-stressed planktonic cells under various growth and cultivation conditions but appears to be irrelevant for UPEC's biofilm formation. IMPORTANCE: Bacterial infections pose an increasing threat to human health exacerbating the demand for alternative treatment options. UPEC, the most common etiological agent of urinary tract infections (UTIs), are confronted by neutrophilic attacks in the bladder, and must therefore be well equipped with powerful defense systems to fend off the toxic effects of RCS. How UPEC deal with the negative consequences of the oxidative burst in the neutrophil phagosome remains unclear. Our study sheds light on the requirements for the expression and protective effects of RcrB, which we recently identified as UPEC's most potent defense system towards HOCl-stress and phagocytosis. Thus, this novel HOCl-stress defense system could potentially serve as an attractive drug target to increase the body's own capacity to fight UTIs.

The identification of a novel protein involved in molybdenum cofactor biosynthesis in Escherichia coli.

In the second step of the molybdenum cofactor (Moco) biosynthesis in Escherichia coli, the l-cysteine desulfurase IscS was identified as the primary sulfur donor for the formation of the thiocarboxylate on the small subunit (MoaD) of MPT synthase, which catalyzes the conversion of cyclic pyranopterin monophosphate to molybdopterin (MPT). Although in Moco biosynthesis in humans, the thiocarboxylation of the corresponding MoaD homolog involves two sulfurtransferases, an l-cysteine desulfurase, and a rhodanese-like protein, the rhodanese-like protein in E. coli remained enigmatic so far. Using a reverse approach, we identified a so far unknown sulfurtransferase for the MoeB-MoaD complex by protein-protein interactions. We show that YnjE, a three-domain rhodanese-like protein from E. coli, interacts with MoeB possibly for sulfur transfer to MoaD. The E. coli IscS protein was shown to specifically interact with YnjE for the formation of the persulfide group on YnjE. In a defined in vitro system consisting of MPT synthase, MoeB, Mg-ATP, IscS, and l-cysteine, YnjE was shown to enhance the rate of the conversion of added cyclic pyranopterin monophosphate to MPT. However, YnjE was not an enhancer of the cysteine desulfurase activity of IscS. This is the first report identifying the rhodanese-like protein YnjE as being involved in Moco biosynthesis in E. coli. We believe that the role of YnjE is to make the sulfur transfer from IscS for Moco biosynthesis more specific because IscS is involved in a variety of different sulfur transfer reactions in the cell.

Redox-Mediated Inactivation of the Transcriptional Repressor RcrR is Responsible for Uropathogenic Escherichia coli's Increased Resistance to Reactive Chlorine Species.

The ability to overcome stressful environments is critical for pathogen survival in the host. One challenge for bacteria is the exposure to reactive chlorine species (RCS), which are generated by innate immune cells as a critical part of the oxidative burst. Hypochlorous acid (HOCl) is the most potent antimicrobial RCS and is associated with extensive macromolecular damage in the phagocytized pathogen. However, bacteria have evolved defense strategies to alleviate the effects of HOCl-mediated damage. Among these are RCS-sensing transcriptional regulators that control the expression of HOCl-protective genes under non-stress and HOCl stress. Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections (UTIs), is particularly exposed to infiltrating neutrophils during pathogenesis; however, their responses to and defenses from HOCl are still completely unexplored. Here, we present evidence that UPEC strains tolerate higher levels of HOCl and are better protected from neutrophil-mediated killing compared with other E. coli. Transcriptomic analysis of HOCl-stressed UPEC revealed the upregulation of an operon consisting of three genes, one of which encodes the transcriptional regulator RcrR. We identified RcrR as a HOCl-responsive transcriptional repressor, which, under non-stress conditions, is bound to the operator and represses the expression of its target genes. During HOCl exposure, however, the repressor forms reversible intermolecular disulfide bonds and dissociates from the DNA resulting in the derepression of the operon. Deletion of one of the target genes renders UPEC significantly more susceptible to HOCl and phagocytosis indicating that the HOCl-mediated induction of the regulon plays a major role for UPEC's HOCl resistance. IMPORTANCE How do pathogens deal with antimicrobial oxidants produced by the innate immune system during infection? Uropathogenic Escherichia coli (UPEC), the most common etiological agent of urinary tract infections (UTIs), is particularly exposed to infiltrating neutrophils and, therefore, must counter elevated levels of the antimicrobial oxidant HOCl to establish infection. Our study provides fundamentally new insights into a defense mechanism that enables UPEC to fend off the toxic effects of HOCl stress. Intriguingly, the defense system is predominantly found in UPEC and absent in noninvasive enteropathogenic E. coli. Our data suggest expression of the target gene rcrB is exclusively responsible for UPEC's increased HOCl tolerance in culture and contributes to UPEC's survival during phagocytosis. Thus, this novel HOCl stress defense system could potentially serve as an attractive drug target to increase the body's own capacity to fight UTIs.

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor.

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein aggregation. The accumulation of protein aggregates in bacterial cells can lead to significant alterations in the cellular phenotypic behavior, including a reduction in growth rates, stress resistance, and virulence. Several experimental procedures exist for the examination of these stressor-mediated phenotypes. This paper describes an optimized assay for the extraction and visualization of aggregated and soluble proteins from different Escherichia coli strains after treatment with a silver-ruthenium-containing antimicrobial. This compound is known to generate reactive oxygen species and causes widespread protein aggregation. The method combines a centrifugation-based separation of protein aggregates and soluble proteins from treated and untreated cells with subsequent separation and visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. This approach is simple, fast, and allows a qualitative comparison of protein aggregate formation in different E. coli strains. The methodology has a wide range of applications, including the possibility to investigate the impact of other proteotoxic antimicrobials on in vivo protein aggregation in a wide range of bacteria. Moreover, the protocol can be used to identify genes that contribute to increased resistance to proteotoxic substances. Gel bands can be used for the subsequent identification of proteins that are particularly prone to aggregation.

Extraction and Quantification of Polyphosphate (polyP) from Gram-negative Bacteria.

Polyphosphate (polyP), a universally conserved biomolecule, is composed of up to 1,000 phosphate monomers linked via phosphoanhydride bonds. Reaching levels in bacteria that are in the high nmoles per mg protein range, polyP plays important roles in biofilm formation and colonization, general stress protection and virulence. Various protocols for the detection of polyP in bacteria have been reported. These methods primarily differ in the ways that polyP is extracted and/or detected. Here, we report an improved method, in which we combine polyP extraction via binding to glassmilk with a very sensitive PolyP kinase/luciferase-based detection system. By using this procedure, we significantly enhanced the sensitivity of polyP detection, making it potentially applicable for mammalian tissues.

Polyphosphate Stabilizes Protein Unfolding Intermediates as Soluble Amyloid-like Oligomers.

Inorganic polyphosphate (polyP) constitutes one of the most conserved and ubiquitous molecules in biology. Recent work in bacteria demonstrated that polyP increases oxidative stress resistance by preventing stress-induced protein aggregation and promotes biofilm formation by stimulating functional amyloid formation. To gain insights into these two seemingly contradictory functions of polyP, we investigated the effects of polyP on the folding model lactate dehydrogenase. We discovered that the presence of polyP during the thermal unfolding process stabilizes folding intermediates of lactate dehydrogenase as soluble micro-ß-aggregates with amyloid-like properties. Size and heterogeneity of the oligomers formed in this process were dependent on the polyP chain length, with longer chains forming smaller, more homogenous complexes. This ability of polyP to stabilize thermally unfolded proteins even upon exposure to extreme temperatures appears to contribute to the observed resistance of uropathogenic Escherichia coli toward severe heat shock treatment. These results suggest that the working mechanism of polyP is the same for both soluble and amyloidogenic proteins, with the ultimate outcome likely being determined by a combination of polyP chain length and the client protein itself. They help to explain how polyP can simultaneously function as general stress-protective chaperone and instigator of amyloidogenic processes in vivo.