Search

Crystalline and porous metal–organic frameworks (MOFs) are potential candidates for different antibacterial, photocatalytic, and adsorption applications. Moreover, multiprincipal element nanoparticles are effective against multidrug‐resistant bacteria, and combining metals with carbon nanomaterials can enhance activity. Herein, a Tri‐MOF comprised of iron, zinc, cobalt and 2‐methyl imidazole is grown together with laser‐induced graphene (LIG) powder. Electron microscopy imaging shows the successful preparation and the crystalline nature of the LIG/Tri‐MOF composite. Fourier‐transform infrared and X‐Ray photoelectron spectroscopy confirm a noncovalent mixture of LIG and Tri‐MOF. Compared with the negligible activity of LIG alone, low doses (0.91–4.54 mg mL−1) of the prepared LIG/Tri‐MOF composite show excellent antibacterial activity (≥95% bacterial removal) and a MIC of 0.6 mg mL−1 for Gram‐negative bacteria, via the gradual leaching of metal ions and organic linker from the material enhanced by bacterial aggregation near the LIG/Tri‐MOF. Compared to a mixture of separately synthesized Tri‐MOF and LIG, the LIG/Tri‐MOF composite shows improved antibacterial effects. All materials show cytotoxicity for L929 mouse cell lines, the solids showing a disrupting effect on cells grown in vitro. Performance‐enhancing combinations of various materials leading to synergistic or additive antimicrobial effects are essential strategies for minimizing the possible emergence of antibiotic‐resistant strains. [ABSTRACT FROM AUTHOR]

Many bacterial pathogens utilize a specialized secretion system, termed type III secretion system (T3SS), to translocate effector proteins into host cells and establish bacterial infection. The T3SS is anchored within the bacterial membranes and contains a long needle/filament that extends toward the host-cell and forms, at its distal end, a pore complex within the host membrane. The T3SS pore complex consists of two bacterial proteins, termed SctB and SctE, which have conflicting targeting indications; a signal sequence that targets to secretion to the extracellular environment via the T3SS, and transmembrane domains (TMDs) that target to membrane localization. In this study, we investigate whether the TMD sequences of SctB and SctE have special features that differentiate them from classical TMDs and allow them to escape bacterial membrane integration. For this purpose, we exchanged the SctB and SctE native TMDs for alternative hydrophobic sequences and found that the TMD sequences of SctB and SctE dictate membrane destination (bacterial versus host membrane). Moreover, we examined the role of the SctB TMD sequence in the activity of the full-length protein, post secretion, and found that the TMD does not serve only as a hydrophobic segment, but is also involved in the ability of the protein to translocate itself and other proteins into and across the host cell membrane. [ABSTRACT FROM AUTHOR]

Diarrheal diseases remain a major public health concern worldwide. Many of the causative bacterial pathogens that cause these diseases have a specialized protein complex, the type III secretion system (T3SS), which delivers effector proteins directly into host cells. These effectors manipulate host cell processes for the benefit of the infecting bacteria. The T3SS structure resembles a syringe anchored within the bacterial membrane, projecting toward the host cell membrane. The entry port of the T3SS substrates, called the export apparatus, is formed by five integral membrane proteins. Among the export apparatus proteins, EscV is the largest, and as it forms a nonamer, it constitutes the largest portion of the export apparatus complex. While there are considerable data on the soluble cytoplasmic domain of EscV, our knowledge of its membrane-associated section and its transmembrane domains (TMDs) is still very limited. In this study, using an isolated genetic reporter system, we found that TMD5 and TMD6 of EscV mediate strong self-oligomerization. Substituting these TMDs within the full-length protein with a random hydrophobic sequence resulted in a complete loss of function of the T3SS, further suggesting that the EscV TMD5 and TMD6 sequences have a functional role in addition to their structural role as membrane anchors. As we observed only mild reduction in the ability of the TMD-exchanged variants to integrate into the full or intermediate T3SS complexes, we concluded that EscV TMD5 and TMD6 are not crucial for the global assembly or stability of the T3SS complex but are rather involved in promoting the necessary TMD–TMD interactions within the complex and the overall TMD orientation to allow channel opening for the entry of T3SS substrates. [ABSTRACT FROM AUTHOR]

The involvement of two polar residues, located in the Escherchia coli aspartate receptor transmembrane (TM) domain, in the dimerization process is investigated. The study demonstrates that different residue combinations in these specific positions have a marked effect on the dimerization ability of the TM domain.

Many gram-negative pathogens utilize a protein complex, termed the type III secretion system (T3SS), to inject virulence factors from their cytoplasm directly into the host cell. An export apparatus that is formed by five putative integral membrane proteins (SctR/S/T/U/V), resides at the center of the T3SS complex. In this study, we characterized the smallest export apparatus protein, SctS, which contains two putative transmembrane domains (PTMD) that dynamically extract from the inner membrane and adopt a helix-turn-helix structure upon assembly of the T3SS. Replacement of each SctS PTMD with an alternative hydrophobic sequence resulted in abolishment of the T3SS activity, yet SctS self- and hetero-interactions as well as the overall assembly of the T3SS complex were unaffected. Our findings suggest that SctS PTMDs are not crucial for the interactions or the assembly of the T3SS base complex but rather that they are involved in adjusting the orientation of the export apparatus relative to additional T3SS sub-structures, such as the cytoplasmic- and the inner-membrane rings. This ensures the fittings between the dynamic and static components of the T3SS and supports the functionality of the T3SS complex. [ABSTRACT FROM AUTHOR]

Type III secretion systems (T3SSs) are central virulence mechanisms used by a variety of Gram-negative bacteria to inject effector proteins into host cells. The needle polymer is an essential part of the T3SS that provides the effector proteins a continuous channel into the host cytoplasm. It has been shown for a fewT3SSs that two chaperones stabilize the needle protein within the bacterial cytosol to prevent its premature polymerization. In this study, we characterized the chaperones of the enteropathogenic Escherichia coli (EPEC) needle protein EscF. We found that Orf2 and Orf29, two poorly characterized proteins encoded within the EPEC locus of enterocyte effacement (LEE), function as the needle protein cochaperones. Our finding demonstrated that both Orf2 and Orf29 are essential for type III secretion (T3S). In addition, we found that Orf2 and Orf29 associate with the bacterial membrane and form a complex with EscF. Orf2 and Orf29 were also shown to disrupt the polymerization of EscF in vitro. Prediction of the tertiary structures of Orf2 and Orf29 showed high structural homology to chaperones of other T3SS needle proteins. Overall, our data suggest that Orf2 and Orf29 function as the chaperones of the needle protein, and therefore, they have been renamed EscE and EscG. [ABSTRACT FROM AUTHOR]

The T3SS (type III secretion system) is a multi-protein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. This apparatus spans both bacterial membranes and transports virulence factors from the bacterial cytoplasm into eukaryotic host cells. The T3SS exports substrates in a hierarchical and temporal manner. The first secreted substrates are the rod/needle proteins which are incorporated into the T3SS apparatus and are required for the secretion of later substrates, the translocators and effectors. In the present study, we provide evidence that rOrf8/EscI, a poorly characterized locus of enterocyte effacement-encoded protein, functions as the inner rod protein of the T3SS of EPEC (enteropathogenic Escherichia coli). We demonstrate that EscI is essential for type III secretion and is also secreted as an early substrate of the T3SS. We found that EscI interacts with EscU, the integral membrane protein that is linked to substrate specificity switching, implicating EscI in the substrate-switching event. Furthermore, we showed that EscI self-associates and interacts with the outer membrane secretin EscC, further supporting its function as an inner rod protein. Overall, the results of the present study suggest that EscI is the YscI/PrgJ/MxiI homologue in the T3SS of attaching and effacing pathogens. [ABSTRACT FROM AUTHOR]

We characterized Orf5 and SepQ, two type III secretion (T3S) system proteins in enteropathogenic Escherichia coli, and showed that they are essential for T3S, associated with the bacterial membrane, and interact with EscN. Our findings suggest that Orf5 and SepQ are homologs of YscL and YscQ from Yersinia, respectively. [ABSTRACT FROM AUTHOR]

Antibiotics are designed to support host defense in controlling infection. Here we describe a paradoxical inhibitory effect of bacteriostatic antibiotics on key mediators of mammalian innate immunity. When growth of species including Escherichia coli and Staphylococcus aureus is suppressed by chloramphenicol or erythromycin, the susceptibility of the bacteria to cathelicidin antimicrobial peptides or serum complement was markedly diminished. Survival of the bacteria in human whole blood, human wound fluid, or a mouse wound infection model was in turn increased after antibiotic-induced bacteriostasis. These findings provide a further rationale against the indiscriminate use of antibiotics. [ABSTRACT FROM AUTHOR]

Examines the role of peptide length and preassembly on the ability of diastereomeric cationic antimicrobial peptides. Expression of similar potent antifungal activity and high antimicrobial activity; Observation of hemolytic activity; Identification of length-dependent antimicrobial activity.

Mentoring has been an efficient tool to help young PIs to deal with the challenges that come with establishing a research groups. Peer Mentoring is an alternative to "classical" mentoring that could benefit female scientists in particular. [ABSTRACT FROM AUTHOR]

Introduction: Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen and one of the major causes of gastrointestinal illness in developing countries. EPEC, similar to many other Gram-negative bacterial pathogens, possesses essential virulence machinery called the type III secretion system (T3SS) that enables the injection of effector proteins from the bacteria into the host cytoplasm. Of these, the translocated intimin receptor (Tir) is the first effector to be injected, and its activity is essential for the formation of attaching and effacing lesions, the hallmark of EPEC colonization. Tir belongs to a unique group of transmembrane domain (TMD)-containing secreted proteins, which have two conflicting destination indications, one for bacterial membrane integration and another for protein secretion. In this study, we examined whether TMDs participate in the secretion, translocation, and function of Tir in host cells., Methods: We created Tir TMD variants with the original or alternative TMD sequence., Results: We found that the C-terminal TMD of Tir (TMD2) is critical for the ability of Tir to escape integration into the bacterial membrane. However, the TMD sequence was not by itself sufficient and its effect was context-dependent. Moreover, the N-terminal TMD of Tir (TMD1) was important for the postsecretion function of Tir at the host cell., Discussion: Taken together, our study further supports the hypothesis that the TMD sequences of translocated proteins encode information crucial for protein secretion and their postsecretion function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Braverman, Gershberg and Sal-Man.)

The type III secretion system (T3SS) is crucial for the virulence of several pathogenic Escherichia coli species as well as for other gram-negative bacterial strains. Therefore, the ability to monitor this system constitutes a valuable tool for assessing the involvement of different proteins in bacterial virulence, for identifying critical domains and specific mutations, and for evaluating the antivirulence activities of various drugs. The major advantage of the T3SS secretion assay for E. coli over assays for other gram-negative pathogens is that it does not necessarily require specific antibodies. Here, we describe how to grow enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) strains under T3SS-inducing conditions, separate the supernatant fraction from the bacterial pellet, analyze this fraction on sodium dodecyl sulfate (SDS)-polyacrylamide gels, and evaluate the level of T3SS activity. We describe a qualitative analysis using Coomassie staining and a quantitative assay using western blotting., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Many Gram-negative bacterial pathogens utilize a specialized protein delivery system, called the type III secretion system (T3SS), to translocate effector proteins into the host cells. The translocated effectors are crucial for bacterial infection and survival. The base of the T3SS transverses both bacterial membranes and contains an export apparatus that comprises five membrane proteins. Here, we study the export apparatus of enteropathogenic Escherichia coli (EPEC) and characterize its central component, called the EscR protein. We found that the third transmembrane domain (TMD) of EscR mediates strong self-oligomerization in an isolated genetic reporter system. Replacing this TMD sequence with an alternative hydrophobic sequence within the full-length protein resulted in a complete loss of function of the T3SS, further suggesting that the EscR TMD3 sequence has another functional role in addition to its role as a membrane anchor. Moreover, we found that an aspartic acid residue, located at the core of EscR TMD3, is important for the oligomerization propensity of TMD3 and that a point mutation of this residue within the full-length protein abolishes the T3SS activity and the ability of the bacteria to translocate effectors into host cells. IMPORTANCE Many Gram-negative bacterial pathogens that cause life-threatening diseases employ a type III secretion system (T3SS) for their virulence. The T3SS comprises several proteins that assemble into a syringe-like structure dedicated to the injection of bacterial virulence factors into the host cells. Although many T3SS proteins are transmembrane proteins, our knowledge of these proteins is limited mostly to their soluble domains. In this study, we found that the third transmembrane domain (TMD) of EscR, a central protein of the T3SS in enteropathogenic E. coli , contributes to protein self-oligomerization. Moreover, we demonstrated that a single aspartic acid residue, located at the core of this TMD, is critical for the activity of the full-length protein and the function of the entire T3SS, possibly due to its involvement in mediating TMD-TMD interactions. Our findings should encourage the mapping of the entire interactome of the T3SS components, including interactions mediated through their TMDs., (Copyright © 2018 Tseytin et al.)

The type III secretion system (T3SS) is a multiprotein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. To ensure that effector proteins are efficiently translocated into the host cell, bacteria must be able to sense their contact with the host cell. In this study, we found that EscP, which was previously shown to function as the ruler protein of the enteropathogenic Escherichia coli T3SS, is also involved in the switch from the secretion of translocator proteins to the secretion of effector proteins. In addition, we demonstrated that EscP can interact with the gatekeeper protein SepL and that the EscP-SepL complex dissociates upon a calcium concentration drop. We suggest a model in which bacterial contact with the host cell is accompanied by a drop in the calcium concentration that causes SepL-EscP complex dissociation and triggers the secretion of effector proteins., Importance: The emergence of multidrug-resistant bacterial strains, especially those of pathogenic bacteria, has serious medical and clinical implications. At the same time, the development and approval of new antibiotics have been limited for years. Recently, antivirulence drugs have received considerable attention as a novel antibiotic strategy that specifically targets bacterial virulence rather than growth, an approach that applies milder evolutionary pressure on the bacteria to develop resistance. A highly attractive target for the development of antivirulence compounds is the type III secretion system, a specialized secretory system possessed by many Gram-negative bacterial pathogens for injecting virulence factors (effectors) into host cells. In this study, we shed light on the molecular mechanism that allows bacteria to sense their contact with the host cell and to respond with the timed secretion of effector proteins. Understanding this critical step for bacterial virulence may provide a new therapeutic strategy., (Copyright © 2017 Shaulov et al.)

Unlabelled: Nontyphoidal Salmonella enterica (NTS) infections are a major burden to global public health, as they lead to diseases ranging from gastroenteritis to systemic infections and there is currently no vaccine available. Here, we describe a highly effective component vaccine against S. enterica serovar Typhimurium in both gastroenteritis and systemic murine infection models. We devised an approach to generate supernatants of S. enterica serovar Typhimurium, an organism that is highly abundant in virulence factors. Immunization of mice with this supernatant resulted in dramatic protection against a challenge with serovar Typhimurium, showing increased survival in the systemic model and decreased intestinal pathology in the gastrointestinal model. Protection correlated with specific IgA and IgG levels in the serum and specific secretory IgA levels in the feces of immunized mice. Initial characterization of the protective antigens in the bacterial culture supernatants revealed a subset of antigens that exhibited remarkable stability, a highly desirable characteristic of an effective vaccine to be used under suboptimal environmental conditions in developing countries. We were able to purify a subset of the peptides present in the supernatants and show their potential for immunization of mice against serovar Typhimurium resulting in a decreased level of colonization. This component vaccine shows promise with regard to protecting against NTS, and further work should significantly help to establish vaccines against these prevalent infections., Importance: Salmonella enterica infections other than typhoid and paratyphoid fever are a major global health burden, as they cause high morbidity and mortality worldwide. Strategies that prevent Salmonella-related diseases are greatly needed, and there is a significant push for the development of vaccines against nontyphoidal Salmonella enterica serovars. In this work, we describe an S. Typhimurium supernatant-derived vaccine that is effective in reducing bacterial colonization in mouse models of gastroenteritis as well as invasive disease. This is a component vaccine that shows high stability to heat, a feature that is important for use under suboptimal conditions, such as those found in sub-Saharan Africa., (Copyright © 2015 Ferreira et al.)

The virulence of many Gram-negative pathogens is associated with type III secretion systems (T3SSs), which deliver virulence effector proteins into the cytoplasm of host cells. Components of enteropathogenic Escherichia coli (EPEC) T3SS are encoded within the locus of enterocyte effacement (LEE). While most LEE-encoded T3SS proteins in EPEC have assigned names and functions, a few of them remain poorly characterized. Here, we studied a small LEE-encoded protein, Orf15, that shows no homology to other T3SS/flagellar proteins and is only present in attaching and effacing pathogens, including enterohemorrhagic E. coli and Citrobacter rodentium. Our findings demonstrated that it is essential for type III secretion (T3S) and that it is localized to the periplasm and associated with the inner membrane. Membrane association was driven by the N-terminal 19 amino acid residues, which were also shown to be essential for T3S. Consistent with its localization, Orf15 was found to interact with the EPEC T3SS outer membrane ring component, EscC, which was previously shown to be embedded within the outer membrane and protruding into the periplasmic space. Interestingly, we found that the predicted coiled-coil structure of Orf15 is critical for the protein's function. Overall, our findings suggest that Orf15 is a structural protein that contributes to the structural integrity of the T3S complex, and therefore we propose to rename it EscA.

The interactions between the TM (transmembrane) domains of many membrane proteins are important for their proper functioning. Mutations of residues into positively charged ones within TM domains were reported to be involved in many genetic diseases, possibly because these mutations affect the self- and/or hetero-assembly of the corresponding proteins. To our knowledge, despite significant progress in understanding the role of various amino acids in TM-TM interactions in vivo, the direct effect of positively charged residues on these interactions has not been studied. To address this issue, we employed the N-terminal TM domain of the aspartate receptor (Tar-1) as a dimerization model system. We expressed within the ToxR TM assembly system several Tar-1 constructs that dimerize via polar- or non-polar amino acid motifs, and mutated these by replacement with a single arginine residue. Our results have revealed that a mutation in each of the motifs significantly reduced the ability of the TMs to dimerize. Furthermore, a Tar-1 construct that contained two arginine residues was unable to correctly integrate itself into the membrane. Nevertheless, an exogenous synthetic Tar-1 peptide containing these two arginine residues was able to inhibit in vivo the marked dimerization of a mutant Tar-1 construct that contained two glutamate residues at similar positions. This indicates that hetero-assembly of TM domains can be mediated by the interaction of two oppositely charged residues, probably by formation of ion pairs. This study broadens our knowledge regarding the effect of positively charged residues on TM-TM interactions in vivo, and provides a potential therapeutic approach to inhibit uncontrolled dimerization of TM domains caused by mutations of polar amino acids.