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Abstract The Omp85 family of outer membrane proteins are ubiquitously distributed among diderm bacteria and play essential roles in outer membrane (OM) biogenesis. The majority of Omp85 orthologs are bipartite and consist of a conserved OM-embedded 16-stranded beta-barrel and variable periplasmic functional domains. Here, we demonstrate that Leptospira interrogans encodes four distinct Omp85 proteins. The presumptive leptospiral BamA, LIC11623, contains a noncanonical POTRA4 periplasmic domain that is conserved across Leptospiraceae. The remaining three leptospiral Omp85 proteins, LIC12252, LIC12254 and LIC12258, contain conserved beta-barrels but lack periplasmic domains. Two of the three ‘noNterm’ Omp85-like proteins were upregulated by leptospires in urine from infected mice compared to in vitro and/or following cultivation within rat peritoneal cavities. Mice infected with a L. interrogans lic11254 transposon mutant shed tenfold fewer leptospires in their urine compared to mice infected with the wild-type parent. Analyses of pathogenic and saprophytic Leptospira spp. identified five groups of noNterm Omp85 paralogs, including one pathogen- and two saprophyte-specific groups. Expanding our analysis beyond Leptospira spp., we identified additional noNterm Omp85 orthologs in bacteria isolated from diverse environments, suggesting a potential role for these previously unrecognized noNterm Omp85 proteins in physiological adaptation to harsh conditions.

Haemophilus parainfluenzae is a Gram-negative opportunist pathogen within the mucus of the nose and mouth without significant symptoms and has an ability to cause various infections ranging from ear, eye, and sinus to pneumonia. A concerning development is the increasing resistance of H. parainfluenzae to beta-lactam antibiotics, with the potential to cause dental infections or abscesses. The principal objective of this investigation is to utilize bioinformatics and immuno-informatic methodologies in the development of a candidate multi-epitope Vaccine. The investigation focuses on identifying potential epitopes for both B cells (B lymphocytes) and T cells (helper T lymphocytes and cytotoxic T lymphocytes) based on high non-toxic and nonallergenic characteristics. The selection process involves identifying human leukocyte antigen alleles demonstrating strong associations with recognized antigenic and overlapping epitopes. Notably, the chosen alleles aim to provide coverage for 90% of the global population. Multi-epitope constructs were designed by using suitable linker sequences. To enhance the immunological potential, an adjuvant sequence was incorporated using the EAAAK linker. The final vaccine construct, comprising 344 amino acids, was achieved after the addition of adjuvants and linkers. This multi-epitope Vaccine demonstrates notable antigenicity and possesses favorable physiochemical characteristics. The three-dimensional conformation underwent modeling and refinement, validated through in-silico methods. Additionally, a protein-protein molecular docking analysis was conducted to predict effective binding poses between the multi-epitope Vaccine and the Toll-like receptor 4 protein. The Molecular Dynamics (MD) investigation of the docked TLR4-vaccine complex demonstrated consistent stability over the simulation period, primarily attributed to electrostatic energy. The docked complex displayed minimal deformation and enhanced rigidity in the motion of residues during the dynamic simulation. Furthermore, codon translational optimization and computational cloning was performed to ensure the reliability and proper expression of the multi-Epitope Vaccine. It is crucial to emphasize that despite these computational validations, experimental research in the laboratory is imperative to demonstrate the immunogenicity and protective efficacy of the developed vaccine. This would involve practical assessments to ascertain the real-world effectiveness of the multi-epitope Vaccine. [ABSTRACT FROM AUTHOR]

The lungs are commonly targeted by Acinetobacter baumannii. The human alveolar basal epithelial cell line, A549, serves as a valuable in vitro model for probing pathogen-cell dynamics. This study examined two Acinetobacter strains, ATCC 19606 and the clinical isolate 58ST, investigating their adherence, internalization, and cytotoxicity within the A549 cell line to illuminate pathogenic mechanisms. Anti-BamA antibodies were expressed, purified, and detected via indirect ELISA. The toxicity of BamA was assessed across BALB/c mice. Both A. baumannii strains were used to infect A549 cells to scrutinize cell invasion diversity. Serum resistance, biofilm creation and inhibition, adhesion, internalization, and intracellular proliferation of live and inactivated A. baumannii were probed with and without anti-BamA sera. A549 cell viability was evaluated in the presence of live A. baumannii and anti-BamA sera-exposed bacteria. Cytoskeleton inhibitor tests were conducted on epithelial cells. A. baumannii strains displayed differing cell invasion aptitudes, with the clinical variant manifesting the highest invasion capability. During internalization, A. baumannii cells localized within vacuoles and migrated towards the nucleus using a zipper-like invasion mechanism. Bacterial division inside host cells culminated in cell demise. Pre-treatment with anti-BamA antibodies substantially impeded A. baumannii's adherence and invasion in epithelial cells. Microscopic imaging validated the intracellular presence of A. baumannii in A549 cells, verifying their invasive potential and residency. These findings substantiate A. baumannii's capacity to proliferate in epithelial cells, with BamA pivotal role against A. baumannii-epithelial cell interplay. This study augments our insight into A. baumannii pathogenesis, facilitating the development of efficacious strategies against A. baumannii infections.

Haemophilus parainfluenzae is a Gram-negative opportunist pathogen within the mucus of the nose and mouth without significant symptoms and has an ability to cause various infections ranging from ear, eye, and sinus to pneumonia. A concerning development is the increasing resistance of H. parainfluenzae to beta-lactam antibiotics, with the potential to cause dental infections or abscesses. The principal objective of this investigation is to utilize bioinformatics and immuno-informatic methodologies in the development of a candidate multi-epitope Vaccine. The investigation focuses on identifying potential epitopes for both B cells (B lymphocytes) and T cells (helper T lymphocytes and cytotoxic T lymphocytes) based on high non-toxic and non-allergenic characteristics. The selection process involves identifying human leukocyte antigen alleles demonstrating strong associations with recognized antigenic and overlapping epitopes. Notably, the chosen alleles aim to provide coverage for 90% of the global population. Multi-epitope constructs were designed by using suitable linker sequences. To enhance the immunological potential, an adjuvant sequence was incorporated using the EAAAK linker. The final vaccine construct, comprising 344 amino acids, was achieved after the addition of adjuvants and linkers. This multi-epitope Vaccine demonstrates notable antigenicity and possesses favorable physiochemical characteristics. The three-dimensional conformation underwent modeling and refinement, validated through in-silico methods. Additionally, a protein-protein molecular docking analysis was conducted to predict effective binding poses between the multi-epitope Vaccine and the Toll-like receptor 4 protein. The Molecular Dynamics (MD) investigation of the docked TLR4-vaccine complex demonstrated consistent stability over the simulation period, primarily attributed to electrostatic energy. The docked complex displayed minimal deformation and enhanced rigidity in the motion of residues during the dynamic simulation. Furthermore, codon translational optimization and computational cloning was performed to ensure the reliability and proper expression of the multi-Epitope Vaccine. It is crucial to emphasize that despite these computational validations, experimental research in the laboratory is imperative to demonstrate the immunogenicity and protective efficacy of the developed vaccine. This would involve practical assessments to ascertain the real-world effectiveness of the multi-epitope Vaccine.

India has a rich history of literature. But it had been governed by the writings of the men and the privileged class for the longest time. The emergence of Dalit writing in Tamil Nadu was a crucial step towards resisting hegemonised dogmas and reclaiming the Dalit identity. Overcoming the shackles of double oppression, Tamil Dalit women came out with works describing their individual and collective experiences of humiliation and rejection. Bama Faustina’s “Karukku” and “Sangati” are two important works that helped bring together the voices of Dalit women through their experiences of trauma and subjugation. Bama explores how caste and religion create a complex web of discrimination and oppression. The novels also highlight the resilience and strength of Dalit women, and their ability to resist and challenge the dominant social order. The intersection of Dalit literature and Western feminism in these works is traced in this article. The works are analysed with the help of feminist theories, to bring out the convergence of gender, patriarchy and caste and class which create a particularly oppressive environment for Dalit women. The article also discusses how third-world feminism paved the way for the representation and acceptance of women in Asian, African and other racial setups.

Bama (born 1958) is considered a significant Dalit woman writer from contemporary India. In her seminal work, Karukku (1992), Bama records her most traumatic experiences in the Convent, Church, seminary, and the Christian community that become the centres of discrimination based on caste. Besides the narratives about the separate human settlement in the village, lower caste people working for upper castes on low wages, the tradition of leftover, hierarchical possession over natural resources like water of lakes, rivers or pastures showcase the mindsets of society. This research paper discusses how Karukku is a Bildungsroman while tracing the growth of the protagonist from childhood into maturity imparting moral and social values through this novel. [ABSTRACT FROM AUTHOR]

A growing aging population is associated with increasing incidences of aging-related diseases and socioeconomic burdens. Hence, research into healthy longevity and aging is urgently needed. Longevity is an important phenomenon in healthy aging. The present review summarizes the characteristics of longevity in the elderly population in Bama, China, where the proportion of centenarians is 5.7-fold greater than the international standard. We examined the impact of genetic and environmental factors on longevity from multiple perspectives. We proposed that the phenomenon of longevity in this region is of high value for future investigations in healthy aging and aging-related disease and may provide guidance for fostering the establishment and maintenance of a healthy aging society. [ABSTRACT FROM AUTHOR]

The outer membrane of Gram-negative bacteria is closely related to the pathogenicity and drug resistance of bacteria. Outer membrane proteins (OMPs) are a class of proteins with important biological functions on the outer membrane. The β-barrel assembly machinery (BAM) complex plays a key role in OMP biogenesis, which ensures that the OMP is inserted into the outer membrane in a correct folding manner and performs nutrient uptake, antibiotic resistance, cell adhesion, cell signaling, and maintenance of membrane stability and other functions. The BAM complex is highly conserved among Gram-negative bacteria. The abnormality of the BAM complex will lead to the obstruction of OMP folding, affect the function of the outer membrane, and eventually lead to bacterial death. In view of the important role of the BAM complex in OMP biogenesis, the BAM complex has become an attractive target for the development of new antibacterial drugs against Gram-negative bacteria. Here, we summarize the structure and function of the BAM complex and review the latest research progress of antibacterial drugs targeting BAM in order to provide a new perspective for the development of antibiotics. [ABSTRACT FROM AUTHOR]

Many Gram-negative bacterial species use contact-dependent growth inhibition (CDI) systems to compete with neighboring cells. CDI+ strains express cell-surface CdiA effector proteins, which carry a toxic C-terminal region (CdiA-CT) that is cleaved from the effector upon transfer into the periplasm of target bacteria. The released CdiA-CT consists of two domains. The C-terminal domain is typically a nuclease that inhibits cell growth, and the N-terminal "cytoplasm-entry" domain mediates toxin translocation into the target-cell cytosol. Here, we use NMR and circular dichroism spectroscopic approaches to probe the structure, stability, and dynamics of the cytoplasm-entry domain from Escherichia coli STEC_MHI813. Chemical shift analysis reveals that the CdiA-CTMHI813 entry domain is composed of a C-terminal helical bundle and a dynamic N-terminal region containing two disulfide linkages. Disruption of the disulfides by mutagenesis or chemical reduction destabilizes secondary structure over the N-terminus, but has no effect on the C-terminal helices. Although critical for N-terminal structure, the disulfides have only modest effects on global thermodynamic stability, and the entry domain exhibits characteristics of a molten globule. We find that the disulfides form in vivo as the entry domain dwells in the periplasm of inhibitor cells prior to target-cell recognition. CdiA-CTMHI813 variants lacking either disulfide still kill target bacteria, but disruption of both bonds abrogates growth inhibition activity. We propose that the entry domain's dynamic structural features are critical for function. In its molten globule-like state, the domain resists degradation after delivery, yet remains pliable enough to unfold for membrane translocation.

ABSTRACT The emergence and spread of antimicrobial resistance (AMR) in Gram-negative pathogens, such as carbapenem-resistant Pseudomonas aeruginosa, pose an increasing threat to health care. Patients with immunodeficiencies or chronic pulmonary disease, like cystic fibrosis (CF), are particularly vulnerable to Pseudomonas infections and depend heavily on antibiotic therapy. To broaden limited treatment options, this study evaluated the potency of the recently licensed drugs ceftazidime-avibactam (CZA), ceftolozane-tazobactam (C/T), and cefiderocol (FDC) as well as two novel preclinical antibiotics, darobactins B (DAR B) and B9 (DAR B9), against clinical P. aeruginosa isolates derived from respiratory samples of CF patients. We observed high levels of resistance to all three newly licensed drugs, with cefiderocol exhibiting the best activity. From the 66 investigated P. aeruginosa isolates, a total of 53% were resistant to CZA, 49% to C/T, and 30% to FDC. Strikingly, 52 of the evaluated isolates were obtained from CF patients prior to market introduction of the drugs. Thus, our results suggest that resistance to CZA, C/T, and FDC may be due to preexisting resistance mechanisms. On the other hand, our two novel preclinical compounds performed better than (CZA and C/T) or close to (FDC) the licensed drugs—most likely due to the novel mode of action. Thus, our results highlight the necessity of global consistency in the area of antibiotic stewardship to prevent AMR from further impairing the potency of antibiotics in clinical practice. Ultimately, this study demonstrates the urgency to support the development of novel antimicrobials, preferably with a new mode of action such as darobactins B and B9, two very promising antimicrobial compounds for the treatment of critically ill patients suffering from multidrug-resistant Gram-negative (MRGN) infections. IMPORTANCE Antimicrobial resistance (AMR) represents an ever increasing threat to the health care system. Even recently licensed drugs are often not efficient for the treatment of infections caused by Gram-negative bacteria, like Pseudomonas aeruginosa, a causative agent of lung infections. To address this unmet medical need, innovative antibiotics, which possess a new mode of action, need to be developed. Here, the antibiogram of clinical isolates derived from cystic fibrosis patients was generated and new bicyclic heptapeptides, which inhibit the outer membrane protein BamA, exhibited strong activity, also against multidrug-resistant isolates.

Contact-dependent growth inhibition (CDI) entails receptor-mediated delivery of CdiA-derived toxins into Gram-negative target bacteria. Using electron cryotomography, we show that each CdiA effector protein forms a filament extending ∼33 nm from the cell surface. Remarkably, the extracellular filament represents only the N-terminal half of the effector. A programmed secretion arrest sequesters the C-terminal half of CdiA, including the toxin domain, in the periplasm prior to target-cell recognition. Upon binding receptor, CdiA secretion resumes, and the periplasmic FHA-2 domain is transferred to the target-cell outer membrane. The C-terminal toxin region of CdiA then penetrates into the target-cell periplasm, where it is cleaved for subsequent translocation into the cytoplasm. Our findings suggest that the FHA-2 domain assembles into a transmembrane conduit for toxin transport into the periplasm of target bacteria. We propose that receptor-triggered secretion ensures that FHA-2 export is closely coordinated with integration into the target-cell outer membrane. VIDEO ABSTRACT.

During the past 40 years of reform and opening-up, China has witnessed great progress in people's health status. Both the occurrence of diseases and people's understanding of health have undergone profound changes. Scholars are focusing on changing socioeconomic factors to maintain health lifestyles, and creating healthy "human–land" relations to prevent diseases. From the perspective of health geography, this research conducted field work in Bama, a longevous area in the Guangxi province in China, and applied the theoretical framework of health lifestyles theory to clarify the construction process of health lifestyles for older people in Bama. The roles and characteristics of different social relations in creating health lifestyles are revealed. The findings of this research can provide a new paradigm for China's health practice research from two aspects: (1) the production of health lifestyles is a knowledge construction process, taking into account the influence of social circumstances, politics, economy, culture, policy, and values; and (2) social attributes affect health lifestyles by reconstructing and improving social relations. [ABSTRACT FROM AUTHOR]

Syphilis is a sexually transmitted infection caused by the highly invasive and immunoevasive spirochetal pathogen Treponema pallidum subsp. pallidum ( TPA ). Untreated syphilis can lead to infection of multiple organ systems, including the central nervous system. The alarming increase in syphilis cases globally underscores the importance of developing novel strategies to understand the complexities of syphilis pathogenesis. In this study, we took advantage of recent advances in in vitro cultivation and genetic manipulation of syphilis spirochetes to engineer a TPA strain that constitutively expresses green fluorescent protein (GFP). GFP + TPA grew identically to the Nichols parent strain in vitro and exhibited wild-type infectivity in the rabbit model. We then used the GFP + strain to visualize TPA interactions with host cells during co-cultivation in vitro , within infected rabbit testes, and following opsonophagocytosis by murine bone marrow-derived macrophages. Development of fluorescent strain also enabled us to develop a flow cytometric-based assay to assess antibody-mediated damage to the spirochete's fragile outer membrane (OM), demonstrating dose-dependent growth inhibition and OM disruption in vitro . Notably, we observed greater OM disruption of GFP + TPA with sera from immune rabbits infected with the TPA Nichols strain compared to sera generated against the genetically distinct SS14 strain. These latter findings highlight the importance of OM protein-specific antibody responses for clearance of TPA during syphilitic infection. The availability of fluorescent TPA strains paves the way for future studies investigating spirochete-host interactions as well as functional characterization of antibodies directed treponemal OM proteins, the presumptive targets for protective immunity., Competing Interests: J.B.P. reports research support from Gilead Sciences and non-financial support from Abbott Laboratories.

This paper aims to analyse two major autobiographies of Dalit women’s literature and African American women’s writing – Karukku (1992) by Bama Faustina and Incidents in the Life of a Slave Girl (1861) by Harriet A. Jacobs – to bring forth the similarities between these two groups of subaltern women. Through the means of autobiography, both writers transmit their own experiences and denounce the gender, race and caste oppression endured. The subaltern theory coined by Antonio Gramsci and developed by Gayatri Spivak will be used to analyse these texts and the way they establish a link between two different worlds as well as how they share the common objective of making their narrators’ exclusion visible in their patriarchal worlds.

The outer membrane of Gram-negative bacteria is closely related to the pathogenicity and drug resistance of bacteria. Outer membrane proteins (OMPs) are a class of proteins with important biological functions on the outer membrane. The β-barrel assembly machinery (BAM) complex plays a key role in OMP biogenesis, which ensures that the OMP is inserted into the outer membrane in a correct folding manner and performs nutrient uptake, antibiotic resistance, cell adhesion, cell signaling, and maintenance of membrane stability and other functions. The BAM complex is highly conserved among Gram-negative bacteria. The abnormality of the BAM complex will lead to the obstruction of OMP folding, affect the function of the outer membrane, and eventually lead to bacterial death. In view of the important role of the BAM complex in OMP biogenesis, the BAM complex has become an attractive target for the development of new antibacterial drugs against Gram-negative bacteria. Here, we summarize the structure and function of the BAM complex and review the latest research progress of antibacterial drugs targeting BAM in order to provide a new perspective for the development of antibiotics.

The article analyzes the migratory chains that constitute South American transvestites and trans women who reside in the Buenos Aires Metropolitan Area during the period 2017-2019, paying special attention to the role of godmothers. The migratory projects of these people are produced because of the hostility and vulnerability they experience in their surroundings and have different instances. One of them is the migratory chains that refer to the social capital necessary to be able to leave the sending society and settle in the receiving society. In this way, South American transvestites/trans women constitute diverse migratory chains that can be characterized as horizontal and vertical, being the godmothers key actors in their diasporas. The focus of the article is qualitative and the method used is grounded theory. According to a theoretical sample consisting of 41 interviews, the information was analyzed using Atlas.Ti. [ABSTRACT FROM AUTHOR]

Transmembrane proteins span lipid bilayer membranes and serve essential functions in all living cells. Membrane-inserted domains are of either α-helical or β-barrel structure. Despite their biological importance, the biophysical mechanisms of the folding and insertion of proteins into membranes are not well understood. While the relative composition of the secondary structure has been examined by circular dichroism spectroscopy in folding studies for several outer membrane proteins, it is currently not known how individual β-strands fold. Here, the folding and insertion of the β-barrel assembly machinery protein A (BamA) from the outer membrane of Escherichia coli into lipid bilayers were investigated, and the formation of strand nine (β9) of BamA was examined. Eight single-cysteine mutants of BamA were overexpressed and isolated in unfolded form in 8 M urea. In each of these mutants, one of the residues of strand β9, from R572 to V579, was replaced by a cysteine and labeled with the fluorophore IAEDANS for site-directed fluorescence spectroscopy. Upon urea-dilution, the mutants folded into the native structure and were inserted into lipid bilayers of dilauroylphosphatidylcholine, similar to wild-type BamA. An aqueous and a membrane-adsorbed folding intermediate of BamA could be identified by strong shifts in the intensity maxima of the IAEDANS fluorescence of the labeled mutants of BamA towards shorter wavelengths, even in the absence of lipid bilayers. The shifts were greatest for membrane-adsorbed mutants and smaller for the inserted, folded mutants or the aqueous intermediates. The spectra of the mutants V573C-, L575C-, G577C-, and V579C-BamA, facing the lipid bilayer, displayed stronger shifts than the spectra recorded for the mutants R572C-, N574C-, T576C-, and K578C-BamA, facing the β-barrel lumen, in both the membrane-adsorbed form and the folded, inserted form. This alternating pattern was neither observed for the IAEDANS spectra of the unfolded forms nor for the water-collapsed forms, indicating that strand β9 forms in a membrane-adsorbed folding intermediate of BamA. The combination of cysteine scanning mutagenesis and site-directed fluorescence labeling is shown to be a valuable tool in examining the local secondary structure formation of transmembrane proteins.

ABSTRACT There is great need for therapeutics against multidrug-resistant, Gram-negative bacterial pathogens. Recently, darobactin A, a novel bicyclic heptapeptide that selectively kills Gram-negative bacteria by targeting the outer membrane protein BamA, was discovered. Its efficacy was proven in animal infection models of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, thus promoting darobactin A as a promising lead compound. Originally discovered from members of the nematode-symbiotic genus Photorhabdus, the biosynthetic gene cluster (BGC) encoding the synthesis of darobactin A can also be found in other members of the class Gammaproteobacteria. Therein, the precursor peptides DarB to -F, which differ in their core sequence from darobactin A, were identified in silico. Even though production of these analogs was not observed in the putative producer strains, we were able to generate them by mutasynthetic derivatization of a heterologous expression system. The analogs generated were isolated and tested for their bioactivity. The most potent compound, darobactin B, was used for cocrystallization with the target BamA, revealing a binding site identical to that of darobactin A. Despite its potency, darobactin B did not exhibit cytotoxicity, and it was slightly more active against Acinetobacter baumannii isolates than darobactin A. Furthermore, we evaluated the plasma protein binding of darobactin A and B, indicating their different pharmacokinetic properties. This is the first report on new members of this new antibiotic class, which is likely to expand to several promising therapeutic candidates. IMPORTANCE Therapeutic options to combat Gram-negative bacterial pathogens are dwindling with increasing antibiotic resistance. This study presents a proof of concept for the heterologous-expression approach to expand on the novel antibiotic class of darobactins and to generate analogs with different activities and pharmacokinetic properties. In combination with the structural data of the target BamA, this approach may contribute to structure-activity relationship (SAR) data to optimize inhibitors of this essential outer membrane protein of Gram-negative pathogens.

The lungs are commonly targeted by Acinetobacter baumannii . The human alveolar basal epithelial cell line, A549, serves as a valuable in vitro model for probing pathogen-cell dynamics. This study examined two Acinetobacter strains, ATCC 19606 and the clinical isolate 58ST, investigating their adherence, internalization, and cytotoxicity within the A549 cell line to illuminate pathogenic mechanisms. Anti-BamA antibodies were expressed, purified, and detected via indirect ELISA. The toxicity of BamA was assessed across BALB/c mice. Both A. baumannii strains were used to infect A549 cells to scrutinize cell invasion diversity. Serum resistance, biofilm creation and inhibition, adhesion, internalization, and intracellular proliferation of live and inactivated A. baumannii were probed with and without anti-BamA sera. A549 cell viability was evaluated in the presence of live A. baumannii and anti-BamA sera-exposed bacteria. Cytoskeleton inhibitor tests were conducted on epithelial cells. A. baumannii strains displayed differing cell invasion aptitudes, with the clinical variant manifesting the highest invasion capability. During internalization, A. baumannii cells localized within vacuoles and migrated towards the nucleus using a zipper-like invasion mechanism. Bacterial division inside host cells culminated in cell demise. Pre-treatment with anti-BamA antibodies substantially impeded A. baumannii 's adherence and invasion in epithelial cells. Microscopic imaging validated the intracellular presence of A. baumannii in A549 cells, verifying their invasive potential and residency. These findings substantiate A. baumannii 's capacity to proliferate in epithelial cells, with BamA pivotal role against A. baumannii -epithelial cell interplay. This study augments our insight into A. baumannii pathogenesis, facilitating the development of efficacious strategies against A. baumannii infections., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The outer membrane of a Gram‐negative bacterium is a crucial barrier between the external environment and its internal physiology. This barrier is bridged selectively by β‐barrel outer membrane proteins (OMPs). The in vivo folding and biogenesis of OMPs necessitates the assistance of the outer membrane chaperone BamA. Nevertheless, OMPs retain the ability of independent self‐assembly in vitro. Hence, it is unclear whether substrate–chaperone dynamics is influenced by the intrinsic ability of OMPs to fold, the magnitude of BamA–OMP interdependence, and the contribution of BamA to the kinetics of OMP assembly. We addressed this by monitoring the assembly kinetics of multiple 8‐stranded β‐barrel OMP substrates with(out) BamA. We also examined whether BamA is species‐specific, or nonspecifically accelerates folding kinetics of substrates from independent species. Our findings reveal BamA as a substrate‐independent promiscuous molecular chaperone, which assists the unfolded OMP to overcome the kinetic barrier imposed by the bilayer membrane. We additionally show that while BamA kinetically accelerates OMP folding, the OMP primary sequence remains a vital deciding element in its assembly rate. Our study provides unexpected insights on OMP assembly and the functional relevance of BamA in vivo. [ABSTRACT FROM AUTHOR]

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Antibiotic resistance has led to an increase in the number of patient hospitalizations and deaths. The situation for gram-negative bacteria is especially dire as the last new class of antibiotics active against these bacteria was introduced to the clinic over 60 years ago, thus there is an immediate unmet need for new antibiotic classes able to overcome resistance. The outer membrane, a unique and essential structure in gram-negative bacteria, contains multiple potential antibacterial targets including BamA, an outer membrane protein that folds and inserts transmembrane β-barrel proteins. BamA is essential and conserved, and its outer membrane location eliminates a barrier that molecules must overcome to access this target. Recently, antibacterial small molecules, natural products, peptides, and antibodies that inhibit BamA activity have been reported, validating the druggability of this target and generating potential leads for antibiotic development. This review will describe these BamA inhibitors, highlight their key attributes, and identify challenges with this potential target. • Transmembrane β-barrel proteins in gram-negative bacterial outer membranes are folded and inserted by the BAM complex • BamA, the central and essential component of the BAM complex, is a potential gram-negative-specific antibacterial target • Small molecules, antibodies, peptides, and natural products that inhibit BAM highlight the druggability of this target • Additional discovery and optimization efforts will be required to move a BamA inhibitor into clinical development [ABSTRACT FROM AUTHOR]

The beta-barrel assembly machinery (BAM) is an indispensable complex for protein transportation located at the outer membrane of bacteria. BAM is composed of five subunits (BamA-E) in the model bacterium Escherichia coli. DR _ 0379 is a BamA homolog in Deinococcus radiodurans , but the other subunits have not been detected in this species. In the present study, deletion of bamA resulted in decreased growth rate and altered morphology of D. radiodurans. ΔbamA cells underwent abnormal cell division, leading to aggregated bacteria of diverse size and shape, and the cell envelope was detached from the cell surface, resulting in reduced resistance to high ionic strength. Oxidative stress resistance was significantly enhanced in the mutant, which may be attributed to increased manganese ion concentration and Mn/Fe ratio. Numerous proteins were released into the medium from ΔbamA cells, including surface layer (S-layer) proteins and various transporters located in the periplasm and outer membrane. These results indicate that BamA affects the synthesis and assembly of the outer membrane and S-layer, and thereby influences material transport and cell division. The findings highlight the special functions of BamA in D. radiodurans , and promote our understanding of the multi-layer structure of the D. radiodurans cell envelope. Unlabelled Image • DR_0379, the BamA homolog of D. radiodurans , is not essential for viability. • Deletion of DR_0379 inhibits the assembly of the outer membrane and the S-layer. • DR_0379 deletion affects growth and cell division in D. radiodurans. • Deletion of DR_0379 changes environmental stress resistance of D. radiodurans. [ABSTRACT FROM AUTHOR]

India has produced many Dalit writers, and Bama stands at the forefront of Dalit literature after publishing her debut touchstone novel Karruku. Later she published yet another significant work Sangati which is also known as a strong Dalit feminine narrative in Tamilnadu. It projects the Dalit women's oppression during the 1960s in Tamilnadu; it also reveals the individual memories of Dalit women and deals with several generations of Dalit women. Sangati in which Bama unveils caste and gender bias faced by the Dalits right from their childhood, discloses the brutal atrocities that happened to children and women in her community, as she pinpoints the double oppression of Dalit women. Hence, this present paper is an attempt to scrutinize the hardships, sufferings, and pain faced by Dalit women right from their childhood, Besides, it also inspects Dalit marginalization, isolation, and dreadful conditions, particularly the tragic conditions of Dalit women in India. The paper investigates how Dalit and Dalit women are exploited in the name of caste. [ABSTRACT FROM AUTHOR]

Protein import into chloroplasts is carried out by the protein translocons at the outer and inner envelope membranes (TOC and TIC). Detailed structures for these translocons are lacking, with only a low‐resolution TOC complex structure available. Recently, we showed that the TOC/TIC translocons can import folded proteins, a rather unique feat for a coupled double membrane system. We also determined the maximum functional TOC/TIC pore size to be 30–35 Å. Here, we discuss how such large pores could form and compare the structural dynamics of the pore‐forming Toc75 subunit to its bacterial/mitochondrial Omp85 family homologs. We put forward structural models that can be empirically tested and also briefly review the pore dynamics of other protein translocons with known structures. [ABSTRACT FROM AUTHOR]

Intestinal colonization by the foodborne pathogen Escherichia coli O157:H7 leads to serious disease symptoms, including hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Synthesis of one or more Shiga toxins (Stx) is essential for HUS and HC development. The genes encoding Stx, including Stx2a, are found within a lambdoid prophage integrated in the E. coli O157:H7 chromosome. Enhanced Stx2a expression was reported when specific non-pathogenic E. coli strains were co-cultured with E. coli O157:H7, and it was hypothesized that this phenotype required the non-pathogenic E. coli to be sensitive to stx-converting phage infection. We tested this hypothesis by generating phage resistant non-pathogenic E. coli strains where bamA (an essential gene and Stx phage receptor) was replaced with an ortholog from other species. Such heterologous gene replacement abolished the ability of the laboratory strain E. coli C600 to enhance toxin production when co-cultured with E. coli O157:H7 strain PA2, which belongs to the hypervirulent clade 8. The extracellular loops of BamA (loop 4, 6, 7) were further shown to be important for infection by stx2a-converting phages. However, similar gene replacement in another commensal E. coli, designated 1.1954, revealed a bamA-independent mechanism for toxin amplification. Toxin enhancement by 1.1954 was not the result of phage infection through an alternative receptor (LamB or FadL), lysogen formation by stx2a-converting phages, or the production of a secreted molecule. Collectively, these data suggest that non-pathogenic E. coli can enhance toxin production through at least two mechanisms.

ABSTRACT Lectin-like bacteriocins (LlpAs) are secreted by proteobacteria and selectively kill strains of their own or related species, and they are composed of two B-lectin domains with divergent sequences. In Pseudomonas spp., initial binding of these antibacterial proteins to cells is mediated by the carboxy-terminal domain through d-rhamnose residues present in the common polysaccharide antigen of their lipopolysaccharide, whereas the amino-terminal domain accounts for strain selectivity of killing. Here, we show that spontaneous LlpA-resistant mutants carry mutations in one of three surface-exposed moieties of the essential β-barrel outer membrane protein insertase BamA, the core component of the BAM complex. Polymorphism of this loop in different Pseudomonas groups is linked to LlpA susceptibility, and targeted cells all share the same signature motif in this loop. Since heterologous expression of such a bamA gene confers LlpA susceptibility upon a resistant strain, BamA represents the primary bacteriocin selectivity determinant in pseudomonads. Contrary to modular bacteriocins that require uptake via the Tol or Ton system, parasitism of BamA as an LlpA receptor advocates a novel bacteriocin killing mechanism initiated by impairment of the BAM machinery. IMPORTANCE Bacteria secrete a variety of molecules to eliminate microbial rivals. Bacteriocins are a pivotal group of peptides and proteins that assist in this fight, specifically killing related bacteria. In Gram-negative bacteria, these antibacterial proteins often comprise distinct domains for initial binding to a target cell’s surface and subsequent killing via enzymatic or pore-forming activity. Here, we show that lectin-like bacteriocins, a family of bacteriocins that lack the prototypical modular toxin architecture, also stand out by parasitizing BamA, the core component of the outer membrane protein assembly machinery. A particular surface-exposed loop of BamA, critical for its function, serves as a key discriminant for cellular recognition, and polymorphisms in this loop determine whether a strain is susceptible or immune to a particular bacteriocin. These findings suggest a novel mechanism of contact-dependent killing that does not require cellular uptake. The evolutionary advantage of piracy of an essential cellular compound is highlighted by the observation that contact-dependent growth inhibition, a distinct antagonistic system, can equally take advantage of this receptor.

In Gram-negative bacteria, the outer membrane contains primarily β-barrel transmembrane proteins and lipoproteins. The insertion and assembly of β-barrel outer-membrane proteins (OMPs) is mediated by the β-barrel assembly machinery (BAM) complex, the core component of which is the 16-stranded transmembrane β-barrel BamA. Recent studies have indicated a possible role played by the seam between the first and last β-barrel strands of BamA in the OMP insertion process through lateral gating and a destabilized membrane region. In this study, we have determined the stability and dynamics of the lateral gate through over 12.5 μs of equilibrium simulations and 4 μs of free-energy calculations. From the equilibrium simulations, we have identified a persistent kink in the C-terminal strand and observed spontaneous lateral-gate separation in a mimic of the native bacterial outer membrane. Free-energy calculations of lateral gate opening revealed a significantly lower barrier to opening in the C-terminal kinked conformation; mutagenesis experiments confirm the relevance of C-terminal kinking to BamA structure and function. [ABSTRACT FROM AUTHOR]

Intestinal colonization by the foodborne pathogen Escherichia coli O157:H7 leads to serious disease symptoms, including hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Synthesis of one or more Shiga toxins (Stx) is essential for HUS and HC development. The genes encoding Stx, including Stx2a, are found within a lambdoid prophage integrated in the E. coli O157:H7 chromosome. Enhanced Stx2a expression was reported when specific non-pathogenic E. coli strains were co-cultured with E. coli O157:H7, and it was hypothesized that this phenotype required the non-pathogenic E. coli to be sensitive to stx-converting phage infection. We tested this hypothesis by generating phage resistant non-pathogenic E. coli strains where bamA (an essential gene and Stx phage receptor) was replaced with an ortholog from other species. Such heterologous gene replacement abolished the ability of the laboratory strain E. coli C600 to enhance toxin production when co-cultured with E. coli O157:H7 strain PA2, which belongs to the hypervirulent clade 8. The extracellular loops of BamA (loop 4, 6, 7) were further shown to be important for infection by stx2a-converting phages. However, similar gene replacement in another commensal E. coli, designated 1.1954, revealed a bamA-independent mechanism for toxin amplification. Toxin enhancement by 1.1954 was not the result of phage infection through an alternative receptor (LamB or FadL), lysogen formation by stx2a-converting phages, or the production of a secreted molecule. Collectively, these data suggest that non-pathogenic E. coli can enhance toxin production through at least two mechanisms. [ABSTRACT FROM AUTHOR]

Dalit literature is a literature of protest, pain, and agony. The primary motive of Dalit literature is the liberation of Dalits. It has become an effective tool in expressing their protest against the domination of the caste Hindus. Its beginning can be traced to the undocumented oral folklore and tales of the past decades. Dalit literature as a genre was established in the 1960's and 1970's when a spurt of Dalit writings was published in Marathi and Gujarati. Dalit literature is also gives importance to the upliftment and liberation of Dalit women from patriarchal and caste ridden society. Dalit women face more atrocities due to caste and gender discrimination. They are subjected to systematic oppression and structural violence both from the general community and from within their own community and their families. Violence is used to curb the assertion of the rights of Dalit women in particular and of the community in general. Their socio-economic status combined with being a woman and Dalit also increase the incidence of violence against them. In Bama's Sangati, Dalit patriarchy is an important subject of concern. Bama criticizes the domestic violence and abuse of Dalit women at home by Dalit men and sexual and occupational harassment faced by them outside their homes at the hands of the upper caste men and the police. The present paper focuses on how Bama's Sangati explores the violence and gender discrimination of Dalit women experienced by the hands of upper caste men and women and their own community male. It is also portrayed, how they are facing these kinds of inhuman treatment to assert their rights and challenge caste and gender norms. [ABSTRACT FROM AUTHOR]

The folding and insertion of integral ß-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect ß-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize amonoclonal antibody that selectively inhibits an essential component of the Escherichia coli ß-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its ß-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outermembrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying ß-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gramnegative bacterial growth by targeting extracellular epitopes. [ABSTRACT FROM AUTHOR]

Untouchability is one of the greatest evils our country has been facing from the time Immemorial. Untouchability is still seen somewhere in direct form and elsewhere in a subdued way. Dalit women are one of the most marginalized segments in the society. The condition of dalit women is more vulnerable than non-dalit women. Dalit women are suffering from multidisadvantages this paper deals with Dalit issues like daily threats of rape, sexual assaults, physical violence at the workplace, in public arena as well as violence at home. [ABSTRACT FROM AUTHOR]

Abstract: Proteins and lipids can form complexes called liprotides, in which the partially denatured protein forms a shell encasing a lipid core. This effectively stabilizes a lipid micelle in an aqueous solvent and suggests that liprotides may provide a suitable vessel for membrane proteins. Accordingly we have investigated if liprotides consisting of α‐lactalbumin and oleate could aid folding of four different outer membrane proteins (OMPs) tOmpA, PagP, BamA, and OmpF. tOmpA was able to fold in the presence of the liprotide, and folding did not occur if only oleate or α‐lactalbumin were added. Although the liprotides did not fold the other three OMPs on its own, it was able to assist their folding in the presence of vesicles. Incubation with liprotides before folding into vesicles increased the folding yield of the outer membrane proteins to a level higher than using micelles of the non‐ionic surfactant DDM. Even though the liprotide was stable at both high urea concentrations and high pH, it failed to efficiently fold OmpA at high pH. Instead, optimal folding was seen at pH 8–9, suggesting that important changes in the liprotide occurred when increasing the pH. We conclude that an otherwise folding‐inactive fatty acid can be activated when presented by a liprotide and thereby work as an in vitro chaperone for outer membrane proteins. [ABSTRACT FROM AUTHOR]

Background: Sub-Saharan Africa has two-thirds of global HIV/AIDS burden. Nigeria has a prevalence of 4.1%, with transmission mainly through heterosexual intercourse. Sixty percent of new infections with HIV occur within the age group 15-29 years and most marriages especially with respect to females are conducted within this age group. Marriages are usually contracted by parents, therefore their knowledge and attitude to HIV status is deemed important. Objective: To assess the knowledge and attitude of parents towards premarital HIV Screening. Method: A cross-sectional descriptive study was conducted among 400 randomly selected parents in three districts of Bama Local Government Area (LGA) using a 36 item structured questionnaire administered to respondents in their homes. Results: A total of 397 respondents (99.3%) have heard of HIV/AIDS, radio (67.3%) being the major source. Sexual intercourse (97.3%), the use of contaminated instruments (42.5%), transmission of infected blood and blood products (17.8%) were identified as modes of transmission. However, only 8.5% identified mother to child transmission. Majority (98.8%) were aware that the disease is preventable mostly through faithfulness to partner (68.8%), premarital abstinence (64.8%) and Condom use (17.5%) while avoidance of contaminated instrument (9%) were also identified as modes of prevention. Majority (96.8%) of parents were aware of premarital HIV Screening. Majority of the parents (97.7%) approve of premarital HIV screening. Many (87.8%) of parents were not aware of the availability of HIV testing facility. Conclusion: The awareness and knowledge of HIV/AIDS in Bama LGA is high among parents. There was also a high level of awareness of PMHS with a positive attitude of parents toward it

Gram-negative bacteria shield themselves from antibiotics by producing an outer membrane (OM) that forms a formidable permeability barrier. Multidrug resistance among these organisms is a particularly acute problem that is exacerbated by the OM. The poor penetrance of many available antibiotics prevents their clinical use, and efforts to discover novel classes of antibiotics against Gram-negative bacteria have been unsuccessful for almost 50 years. Recent insights into how the OM is built offer new hope. Several essential multiprotein molecular machines (Bam, Lpt, and Lol) work in concert to assemble the barrier and offer a swathe of new targets for novel therapeutic development. Murepavadin has been at the vanguard of these efforts, but its recently reported phase III clinical trial toxicity has tempered the anticipation of imminent new clinical options. Nonetheless, the many concerted efforts aimed at breaking down the OM barrier provide a source of ongoing optimism for what may soon come through the development pipeline. We will review the current state of drug development against the OM assembly targets, highlighting insightful new discovery approaches and strategies.

Pyogenic hepatic abscess (PHA) is a rare, but potentially serious disease. At present, ultrasound-guided or computed tomography-guided percutaneous needle aspiration or catheter drainage is appropriate as a first-line treatment. However, it is difficult to aspirate or drain pus and to select the appropriate antibiotic therapy if the abscess consists of thick pus and polymicrobial confections, or its pathogenic bacterium is multidrug resistant and cryptogenic. Case studies of alcoholization provide a novel method to manage PHA. However, the efficacy and safety of this treatment should be further evaluated. In the present study, the therapeutic efficacy and complications of alcoholization for PHAs in Bama minipigs were investigated. PHAs were prospectively treated by ultrasound-guided percutaneous instillation of absolute alcohol in the abscess cavity. The criteria for considering a successful intervention were met in all minipigs subsequent to alcoholization twice within 14 days. The procedures were well tolerated in all animals, and there were no alcoholic adverse effects or procedure-associated complications. In conclusion, ultrasound-guided percutaneous alcoholization is a safe and effective procedure to manage PHA. The problems of thick pus aspiration and selection of an appropriate antibiotic observed in other treatments were resolved effectively using alcoholization. This technique may reduce the treatment period and possibly become a novel strategy for the management of PHA. [ABSTRACT FROM AUTHOR]

Summary The β-barrel assembly machine (BAM) mediates the biogenesis of outer membrane proteins (OMPs) in Gram-negative bacteria. BamA, the central BAM subunit composed of a transmembrane β-barrel domain linked to five polypeptide transport-associated (POTRA) periplasmic domains, is thought to bind nascent OMPs and undergo conformational cycling to catalyze OMP folding and insertion. One model is that conformational flexibility between POTRA domains is part of this conformational cycling. Nuclear magnetic resonance (NMR) spectroscopy was used here to study the flexibility of the POTRA domains 1–5 in solution. NMR relaxation studies defined effective rotational correlational times and together with residual dipolar coupling data showed that POTRA1–2 is flexibly linked to POTRA3–5. Mutants of BamA that restrict flexibility between POTRA2 and POTRA3 by disulfide crosslinking displayed impaired function in vivo. Together these data strongly support a model in which conformational cycling of hinge motions between POTRA2 and POTRA3 in BamA is required for biological function. [ABSTRACT FROM AUTHOR]

Bama, originally called Faustina Mary Fathima Rani is a Paraiyar, Christian Dalit activist. Her novels focus on caste and gender discrimination. They portray caste-discrimination practised in Christianity and Hinduism. Bama's works are seen as embodying Dalit feminism and are famed for celebrating the inner strength of the subaltern woman. Bama rose to fame with her autobiographical novel Karukku (1992), which chronicles the joys and sorrows experienced by Dalit Christian women in Tamilnadu. Bama's Karukku (1992) and Sangati (1994) narrate the painful and tormented life history of a sensitive, insightful and perceptive Dalit woman. The word 'sangati' means 'events' and thus the novel, through individual stories, anecdotes and memoirs, portrays the events that take place in the life of women in the Paraiyar community. The novel also reveals how the Paraiyar women are doubly oppressed. Bama's Karukku and Sangati show various aspects of caste discrimination and gender discrimination. These novels show the sufferings of Dalit community in the hands of "upper-caste" communities in church, places of education and society. These also show the sufferings of the Dalit women in the hands of the upper caste men and also in the hands of their own husbands. [ABSTRACT FROM AUTHOR]