Enterococcus faecalis is a Gram-positive bacterium that is a major cause of hospital-acquired infections, in part due to its intrinsic resistance to cephalosporins. The mechanism that confers intrinsic cephalosporin resistance in enterococci remains incompletely defined. Previously, we have shown that the Ser/Thr protein kinase and phosphatase pair IreK and IreP act antagonistically to regulate cephalosporin resistance in E. faecalis. We hypothesize that IreK senses antibiotic-induced cell wall damage and activates a signaling pathway leading to antibiotic resistance. However, the factors downstream of IreK have not yet been identified. To discover such factors, suppressor mutations that restored resistance to a ⌬ireK kinase mutant were identified. Mutations were found in IreB, a highly conserved gene of unknown function that is widespread among low-GC Gram-positive bacteria. We show that IreB plays a negative regulatory role in cephalosporin resistance and is an endogenous substrate of both IreK and IreP. IreB is phosphorylated on conserved threonine residues, and mutations at these sites impair cephalosporin resistance. Our results are consistent with a model in which the activity of IreB is modulated by IreK-dependent phosphorylation in a signaling pathway required for cephalosporin resistance and begin to shed light on the function of this previously uncharacterized protein.

Antibiotic-resistant enterococci are major causes of hospital-acquired infections and therefore represent a serious public health problem. One well-known risk factor for the acquisition of hospital-acquired enterococcal infections is prior therapy with broad-spectrum cephalosporin antibiotics. Enterococci can proliferate in patients undergoing cephalosporin therapy due to intrinsic cephalosporin resistance, a characteristic of the genus Enterococcus. However, the molecular basis for cephalosporin resistance in E. faecalis has yet to be adequately elucidated. Previously we determined that a putative Ser/Thr kinase, IreK (formerly PrkC), is required for intrinsic cephalosporin resistance in E. faecalis. Here we show that kinase activity is required for cephalosporin resistance and, further, that resistance in E. faecalis is reciprocally regulated by IreK and IreP, a PP2C-type protein phosphatase encoded immediately upstream of IreK. Mutants of two divergent lineages of E. faecalis lacking IreP exhibit remarkable hyperresistance to cephalosporins but not to antibiotics targeting other cellular processes. Further genetic analyses indicate that hyperresistance of the IreP mutant is mediated by the IreK kinase. Additionally, competition experiments reveal that hyperresistant ΔireP mutants exhibit a substantial fitness defect in the absence of antibiotics, providing an evolutionary rationale for the use of a complex signaling system to control intrinsic cephalosporin resistance. These results support a model in which IreK and IreP act antagonistically via protein phosphorylation and dephosphorylation as part of a signal transduction circuit to regulate cellular adaptation to cephalosporin-induced stress.

Botulinum neurotoxin (BoNT; serotypes A-G) and tetanus neurotoxin elicit flaccid and spastic paralysis, respectively. These neurotoxins are zinc proteases that cleave SNARE proteins to inhibit synaptic vesicle fusion to the plasma membrane. Although BoNT/B and tetanus neurotoxin (TeNT) cleave VAMP-2 at the same scissile bond, their mechanism(s) of VAMP-2 recognition is not clear. Mapping experiments showed that residues 60 -87 of VAMP-2 were sufficient for efficient cleavage by BoNT/B and that residues 40 -87 of VAMP-2 were sufficient for efficient TeNT cleavage. Alanine-scanning mutagenesis and kinetic analysis identified three regions within VAMP-2 that were recognized by BoNT/B and TeNT: residues adjacent to the site of scissile bond cleavage (cleavage region) and residues located within N-terminal and C-terminal regions relative to the cleavage region. Analysis of residues within the cleavage region showed that mutations at the P7, P4, P2, and P1 residues of VAMP-2 had the greatest inhibition of LC/B cleavage (>32-fold), whereas mutations at P7, P4, P1, and P2 residues of VAMP-2 had the greatest inhibition of LC/TeNT cleavage (>64-fold). Residues within the cleavage region influenced catalysis, whereas residues N-terminal and C-terminal to the cleavage region influenced binding affinity. Thus, BoNT/B and TeNT possess similar organization but have unique residues to recognize and cleave VAMP-2. These studies provide new insights into how the clostridial neurotoxins recognize their substrates.The botulinum neurotoxins (BoNTs) 2 are the most potent protein toxins for humans (1). BoNTs have been used for malicious purposes (2) but also relieve numerous human neurological afflictions, such as blepharospasm, in addition to being used for cosmetic enhancement (3-5). Recent studies provide a more detailed understanding of BoNT action, which should provide insight for developing novel products and therapies to treat human suffering.BoNTs are zinc proteases that cleave SNARE proteins to block synaptic vesicle fusion and neurotransmitter release to elicit flaccid paralysis. BoNTs are 150-kDa dichain proteins with AB structure-function properties; the N terminus encodes the zinc protease domain (light chain (LC)), and the C terminus encodes a translocation domain and a receptor binding domain (6, 7). The seven serotypes of BoNTs, termed A-G, are based upon antisera neutralization properties (8). Each BoNT serotype cleaves one of three neuronal SNARE proteins: SNAP-25, VAMP-2, or syntaxin 1a, except for BoNT/C, which cleaves both SNAP-25 and syntaxin 1a. BoNT/A and BoNT/E cleave SNAP-25 at different sites, whereas BoNT/B, BoNT/D, BoNT/F, and BoNT/G cleave VAMP-2 at distinct sites (9 -12). In addition, tetanus neurotoxin (TeNT) shares structural homology with the BoNTs and cleaves VAMP-2 at the same site as BoNT/B. Despite this similarity, TeNT elicits spastic paralysis due to the ability to retrograde traffic within the peripheral neurons to target interneurons (6, 13-15).Unlike other zinc proteases, BoNTs and TeNT rec...

Signaling pathways allow bacteria to adapt to changing environments. For pathogenic bacteria, signaling pathways allow for timely expression of virulence factors and the repression of antivirulence factors within the mammalian host. As the bacteria exit the mammalian host, signaling pathways enable the expression of factors promoting survival in the environment and/or nonmammalian hosts. One such signaling pathway uses the dinucleotide cyclic-di-GMP (c-di-GMP), and many bacterial genomes encode numerous proteins that are responsible for synthesizing and degrading c-di-GMP. Once made, c-di-GMP binds to individual protein and RNA receptors to allosterically alter the macromolecule function to drive phenotypic changes. Each bacterial genome encodes unique sets of genes for c-di-GMP signaling and virulence factors so the regulation by c-di-GMP is organism specific. Recent works have pointed to evidence that c-di-GMP regulates virulence in different bacterial pathogens of mammalian hosts. In this review, we discuss the criteria for determining the contribution of signaling nucleotides to pathogenesis using a well-characterized signaling nucleotide, cyclic AMP (cAMP), in Pseudomonas aeruginosa. Using these criteria, we review the roles of c-di-GMP in mediating virulence and highlight common themes that exist among eight diverse pathogens that cause different diseases through different routes of infection and transmission. © 2017 Wiley Periodicals, Inc. How to cite this article:WIREs RNA 2018, 9:e1454. doi: 10.1002/wrna.1454 INTRODUCTIONB is-(3 0 -5 0 )-cyclic dimeric guanosine monophosphate (c-di-GMP) is a widely utilized signaling pathway that regulates bacterial adaptation to different environments. 1 C-di-GMP is synthesized from two GTP by diguanylate cyclases (DGCs) that contain a GGDEF domain and degraded into pGpG by phosphodiesterases (PDE-As) that contain EAL or HD-GYP domain. Signaling by c-di-GMP is completed when pGpG is degraded further to GMP by oligoribonuclease and related RNA degradation enzymes. [2][3][4] Thus, c-di-GMP represents one of the shortest signaling ribonucleotides. In addition, a number of genes can encode proteins with both GGDEF and EAL domains (GGDEF-EAL), indicating that levels of c-di-GMP in the cell are regulated by a complicated number of synthetic and degradative enzymes. Once made, c-di-GMP binds a diverse set of protein receptors and RNA riboswitches. 1 C-di-GMP binding to molecular receptors allosterically alters their function and downstream cellular phenotypes. 5 Genes encoding c-di-GMP signaling components are found in a large number of bacterial pathogens. This review seeks to cover the contribution of c-di-GMP signaling to bacterial pathogenesis for a number of different pathogens to identify common themes. For this review, virulence and pathogenesis is considered as the ability of the bacteria to replicate and cause disease symptoms in the mammalian host. Owing to space limitation, for each pathogen, we have referenced reviews that cover their virulence factors and their c...

dBrucella melitensis is a well-adapted zoonotic pathogen considered a scourge of mankind since recorded history. In some cases, initial infection leads to chronic and reactivating brucellosis, incurring significant morbidity and economic loss. The mechanism by which B. melitensis subverts adaptive immunological memory is poorly understood. Previous work has shown that Brucellaspecific CD8؉ T cells express gamma interferon (IFN-␥) and can transition to long-lived memory cells but are not polyfunctional. In this study, chronic infection of mice with B. melitensis led to CD8 ؉ T cell exhaustion, manifested by programmed cell death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) expression and a lack of IFN-␥ production. ؉ T cells increased after adoptive transfer in both challenged and unchallenged recipients. CD8 ؉ T cells of challenged recipients initially retained the stunted IFN-␥ production found prior to transfer, and cells from acutely infected mice were never seen to transition to either memory subset at all time points tested, up to 30 days post-primary infection, suggesting a delay in the generation of memory. Here we have identified defects in Brucella-responsive CD8 ؉ T cells that allow chronic persistence of infection. Brucellosis caused by Brucella melitensis has a high incidence in developing countries, and the World Health Organization considers brucellosis one of the seven neglected zoonoses, a group of diseases that contribute to the perpetuation of poverty (1, 2). Brucella has many mechanisms to survive and replicate in hostile host cells, including inducing the unfolded-protein response (UPR), hijacking host nutrients, and counteracting the effects of pH changes, among many others (3-6). The chronic, reactivating nature of Brucella infection, along with its stealthy intracellular life-style, makes infections difficult to clear and requires lengthy antibiotic treatment (7-9). CD8 ϩ T cells control intracellular infections by identifying and killing compromised host cells as a part of the adaptive immune response (10, 11). Recognition of nonself antigenic epitopes in the context of major histocompatibility complex (MHC) class I by cytotoxic T cells also leads to the release of effector molecules to increase local inflammation, thereby "raising the alert" of the host in response to intracellular infection (12). A subset of MHC class I-restricted epitopes of Brucella melitensis generated during infection has been characterized and can elicit specific CD8 ϩ T cells (13). These T cells have been shown to kill their target cells, release cytokines, and survive into the chronic phase of infection (7). Why, then, in the successful establishment of chronic brucellosis, do we see the highly evolved CD8 ϩ T cell arm of adaptive immunity fail to protect the host from long-term infection?Immunological memory is the ability of the host to mount a fast, effective secondary response to infection. CD8 ϩ T cell memory is derived from effectors generated during primary infection or vaccination, a small cohort of ...

f Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host-Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a ⌬bpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the ⌬bpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase ⌬cgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, ⌬bpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection. Brucella species are Gram-negative, facultative intracellular bacterial pathogens that cause brucellosis, the most prevalent zoonosis worldwide (1, 2). With more than 500,000 infections per year, the high incidence of brucellosis in southeastern Europe, the Mediterranean, South America, and Africa causes a major economic burden (2, 3). In animals, brucellosis is characterized by increased abortion, weak offspring, and decreased milk production. Brucella melitensis is the predominant cause of human brucellosis; however, B. abortus, B. suis, and B. canis can also infect humans (4). Human brucellosis is typically acquired by consuming contaminated milk products or via inhalation of aerosolized bacteria from occupational hazards (5). Human brucellosis is a debilitating disease in which most people initially experience a period of undulating fever which can progress to a chronic infection if untreated or if antibiotic treatment fails. Complications of chronic infections include liver damage, orchitis, endocarditis, and arthritis (1, 4).Brucella spp. have the ability to infect both professional and nonprofessional phagocytes (6). Because of this, Brucella spp. encounter varied environments both throughout the body and within a cell and must adapt accordingly. To date, few virulence factors have been identified in Brucella, and even less is known about how these virulence factors are regulated. Subsequently, little is known how Brucella adapts to its rapidly changing environments and how it alternates between acute and chronic virulence.The second messenger cyclic di-GMP (c-di-GMP...

Pseudomonas aeruginosa is an opportunistic pathogen that affects a large proportion of cystic fibrosis (CF) patients. CF patients have dehydrated mucus within the airways that leads to the inability of the mucociliary escalator to expel inhaled microbes. Once inhaled, P. aeruginosa can persist in the lungs of the CF patients for the remainder of their lives. During this chronic infection, a phenomenon called mucoid conversion can occur in which P. aeruginosa can mutate and inactivate their mucA gene. As a consequence, transcription of the alg operon is highly expressed, leading to the copious secretion of the alginate exopolysaccharide, which is associated with decreased lung function and increased CF patient morbidity and mortality. Alginate biosynthesis by P. aeruginosa is post-translationally regulated by bis(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), which binds to the receptor protein Alg44 to activate alginate production. The identification of small molecules that disrupt the binding of c-di-GMP to Alg44 could inhibit the ability of P. aeruginosa to produce alginate. In this work, a class of thiol-benzo-triazolo-quinazolinone compounds that inhibited Alg44 binding to c-di-GMP in vitro was identified after screening chemical libraries consisting of ∼50 000 chemical compounds. Thiol-benzo-triazolo-quinazolinones were shown to specifically inhibit Alg44-c-di-GMP interactions by forming a disulfide bond with the cysteine residue in the PilZ domain of Alg44. The more potent thiol-benzo-triazolo-quinazolinone had the ability to reduce P. aeruginosa alginate secretion by up to 30%. These compounds serve as leads in the development of novel inhibitors of alginate production by P. aeruginosa after mucoid conversion.

Chronic bacterial infections on medical devices, including catheter-associated urinary tract infections (CAUTI), are associated with bacterial biofilm communities that are refractory to antibiotic therapy and resistant to host immunity. Previously, we have shown that Pseudomonas aeruginosa can cause CAUTI by forming a device-associated biofilm that is independent of known biofilm exopolysaccharides. Here, we show by RNA-seq that host urine alters the transcriptome of P. aeruginosa by suppressing quorum sensing regulated genes. P. aeruginosa produces acyl homoserine lactones (AHLs) in the presence of urea, but cannot perceive AHLs. Repression of quorum sensing by urine implies that quorum sensing should be dispensable during infection of the urinary tract. Indeed, mutants defective in quorum sensing are able to colonize similarly to wild-type in a murine model of CAUTI. Quorum sensing-regulated processes in clinical isolates are also inhibited by urea. These data show that urea in urine is a natural anti-quorum sensing mechanism in mammals.