Bacterial Internalization Research Articles

Cellulosic schiff base hydrogel biosensor for bacterial detection with pH/thermo-responsitivity: DFT calculations and molecular docking

A facile green hydrothermal method was developed for the preparation of fluorescent carbon dots (CDs) using sugarcane bagasse (SB) as a raw material. The incorporation of CDs within the hydroxyethyl cellulose-acrylamide hydrogel (HEC-AM) in the presence of glutardehyde (GA) as a crosslinking agent showed bright red emission under fluorescence microscope. The hydroxyethyl cellulose-N–CDs-acrylamide hydrogel (HEC-AM@N–CDs) were used as alternative biocompatible fluorescent probes for imaging and suppression of Gram-negative bacteria such as Escherichia coli (with inhibition zones 13 mm for HEC-AM to 17 mm for HEC-AM@N–CDs) &Helicobacter pylori (with inhibition zones 16 mm for HEC-AM to 13 mm for HEC-AM@N–CDs) and Gram-positive bacteria such as Micrococcus luteus (with inhibition zones 15 mm for HEC-AM to 16 mm for HEC-AM@N–CDs) &Staphylococcus aureus (with inhibition zones 13 mm for HEC-AM to 14 mm for HEC-AM@N–CDs). The N–CDs promote bacterial binding and internalization, leading to differential fluorescence emission depending on the bacteria's cell wall composition. DFT calculations support strong interactions between the bacteria and HEC-AM@N–CDs.

Characterizing the interplay between Acinetobacter baumannii, A549 cells, and anti-Omp34 antibodies: implications for adherence, internalization, and cytotoxicity.

Acinetobacter baumannii thrives within eukaryotic cells, influencing persistence, treatment approaches, and progression of disease. We probed epithelial cell invasion by A. baumannii and the influence of antibodies raised to outer membrane protein 34 (Omp34) on epithelial interactions. We expressed and purified recombinant Omp34 and induced anti-Omp34 antibodies in Bagg albino or BALB/c mice. Omp34 was evaluated for acute toxicity in mice through histological analysis of six organs. The host cell line, A549, was exposed to both A. baumannii 19606 and a clinical isolate. The study also investigated serum resistance, adherence, internalization, and proliferation of A. baumannii in A549 cells, with and without anti-Omp34 sera, utilizing cell culture techniques and light microscopy. A549 cell viability was evaluated by A. baumannii challenge and exposure to anti-Omp34 sera. Actin disruption experiments using cytochalasin D probed microfilament and microtubule roles in A. baumannii invasion. Omp34 prompted antibody production without toxicity in mice. The serum showed bactericidal effects on both strains. Additionally, both A. baumannii strains were found to form biofilms. Omp34 serum was observed to decrease biofilm formation, bacterial adherence, internalization, and proliferation in A549 cells. Furthermore, the use of anti-Omp34 serum enhanced the post-infection survival of the host cell. Pre-exposure of A549 cells to cytochalasin D reduced bacterial internalization, highlighting the role of actin polymerization in the invasion process. Microscopic analysis revealed various interactions, such as adherence, membrane alterations, vacuolization, apoptosis, and cellular damage. Anti-Omp34 serum-exposed A549 cells were protected and showed reduced damage. The findings reveal that A. baumannii can significantly multiply intracellularly within host cells. This suggests the bacterium's ability to establish an environment conducive to its replication by preventing fusion with degradative lysosomes and inhibiting acidification. This finding contributes to the understanding of A. baumannii's intracellular persistence and highlights the role of Omp34 in influencing apoptosis, autophagy, and bacterial adherence, which may impact the development of effective treatments against A. baumannii infections.

Medulla Tetrapanacis water extract ameliorates mastitis by suppressing bacterial internalization and inflammation via MAPKs signaling in vitro and in vivo

AbstractMedulla Tetrapanacis (MT) is a commonly used herbal ingredient in soup to promote lactation and management of mastitis among lactating mothers worldwide, particularly in Asian countries. However, scientific evidence to support its usage in the management of mastitis is limited. This study aimed to investigate the effects of MT water extract and its underlying mechanisms in Staphylococcus aureus (SA)–induced mastitis in human mammary epithelial cells (HuMEC) and lipopolysaccharide (LPS)–induced mastitis in lactating Sprague–Dawley (SD) rats. The anti‐inflammatory and antibacterial effects of MT water extract were examined in SA‐infected HuMEC by ELISA and plate counting, respectively. The effects of MT water extract on blood‐milk barrier and the underlying mechanism of action of the MT water extract were also investigated by transendothelial electrical resistance assay and western blot. The effects and mechanisms of MT water extract on alleviating mastitis on SD rats were evaluated using LPS‐induced mastitis rat model. Our results showed that MT water extract could suppress SA‐induced mastitis by reducing SA internalization and growth, protecting blood‐milk barrier integrity, and attenuating release of cytokines (interleukin 6 [IL‐6] and tumor necrosis factor‐alpha [TNF‐α]) in HuEMC. Furthermore, the antibacterial effect might be related to the increase of antimicrobial peptides transcription, and the anti‐inflammatory effect is at least partly mediated by inactivation of p38 and JNK signaling pathways. Finally, our in vivo results showed that MT water extract ameliorated LPS‐induced mastitis in SD rats via suppressing inflammatory cytokine release (TNF‐α, IL‐6, and interleukin‐1 beta), myeloperoxidase, and alleviating pathohistological damage by downregulation of JNK signaling pathway.

Alpha-hemolysin promotes internalization of Staphylococcus aureus into human lung epithelial cells via caveolin-1- and cholesterol-rich lipid rafts

Staphylococcus aureus is a pathogen associated with severe respiratory infections. The ability of S. aureus to internalize into lung epithelial cells complicates the treatment of respiratory infections caused by this bacterium. In the intracellular environment, S. aureus can avoid elimination by the immune system and the action of circulating antibiotics. Consequently, interfering with S. aureus internalization may represent a promising adjunctive therapeutic strategy to enhance the efficacy of conventional treatments. Here, we investigated the host-pathogen molecular interactions involved in S. aureus internalization into human lung epithelial cells. Lipid raft-mediated endocytosis was identified as the main entry mechanism. Thus, bacterial internalization was significantly reduced after the disruption of lipid rafts with methyl-β-cyclodextrin. Confocal microscopy confirmed the colocalization of S. aureus with lipid raft markers such as ganglioside GM1 and caveolin-1. Adhesion of S. aureus to α5β1 integrin on lung epithelial cells via fibronectin-binding proteins (FnBPs) was a prerequisite for bacterial internalization. A mutant S. aureus strain deficient in the expression of alpha-hemolysin (Hla) was significantly impaired in its capacity to enter lung epithelial cells despite retaining its capacity to adhere. This suggests a direct involvement of Hla in the bacterial internalization process. Among the receptors for Hla located in lipid rafts, caveolin-1 was essential for S. aureus internalization, whereas ADAM10 was dispensable for this process. In conclusion, this study supports a significant role of lipid rafts in S. aureus internalization into human lung epithelial cells and highlights the interaction between bacterial Hla and host caveolin-1 as crucial for the internalization process.

Bactericidal ability of target acidic phospholipids and phagocytosis of CDC42 GTPase-mediated cytoskeletal rearrangement underlie functional conservation of CXCL12 in vertebrates.

Chemokine CXCL12 plays a crucial role in both direct bactericidal activity and phagocytosis in humans. However, the mechanisms and evolutionary functions of these processes in vertebrates remain largely unknown. In this study, we found that the direct bactericidal activity of CXCL12 is highly conserved across various vertebrate lineages, including Arctic lamprey (Lampetra japonica), Basking shark (Cetorhinus maximus), grass carp (Ctenopharyngodon idella), Western clawed frog (Xenopus tropicalis), Green anole (Anolis carolinensis), chicken (Gallus gallus), and human (Homo sapiens). CXCL12 also has been shown to promote phagocytosis in lower and higher vertebrates. We then employed C. idella CXCL12a (CiCXCL12a) as a model to further investigate its immune functions and underlying mechanisms. CiCXCL12a exerts direct broad-spectrum antibacterial activity by targeting bacterial acidic phospholipids, resulting in bacterial cell membrane perforation, and eventual lysis. Monocytes/macrophages are attracted to the infection sites for phagocytosis through the rapid production of CiCXCL12a during bacterial infection. CiCXCL12a induces CDC42 and CDC42 GTPase activation, which in turn mediates F-actin polymerization and cytoskeletal rearrangement. The interaction between F-actin and Aeromonas hydrophila facilitates bacterial internalization into monocytes/macrophages. Additionally, A. hydrophila is colocalized within early endosomes, late endosomes and lysosomes, ultimately degrading within phagolysosomes. CiCXCL12a also activates PI3K-AKT, JAK-STAT5 and MAPK-ERK signaling pathways. Notably, only the PI3K-AKT signaling pathway inhibits LPS-induced monocyte/macrophage apoptosis. Thus, CiCXCL12a plays key roles in reducing tissue bacterial loads, attenuating organ injury, and decreasing mortality rates. Altogether, our findings elucidate the conserved mechanisms underlying CXCL12-mediated bactericidal activity and phagocytosis, providing novel perspectives into the immune functions of CXCL12 in vertebrates.

Enhancing safe and effective treatment of carbapenem-resistant Klebsiella pneumoniae with polymyxin B-loaded dendritic nanoparticles

Polymyxin B (PMB) is highly effective against Gram-negative bacterial infections, notably in targeting lipopolysaccharide (LPS), making it a vital last-resort treatment for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. However, its clinical application is hampered by nephrotoxicity and complications arising from its positive charge. This study introduces dendritic mesoporous organosilica nanoparticles loaded with PMB (PBSi-NPs), designed to overcome these limitations. The PBSi-NPs, with an average size of 170 nm, demonstrate effective LPS scavenging, enhanced bacterial internalization, and potent bactericidal properties. Their ability to release PMB in acidic microenvironments, characteristic of bacterial infections, significantly enhances the management of severe CRKP-induced infections. Notably, PBSi-NPs exhibit reduced toxicity towards human embryonic kidney cells (HEK 293), attributed to their stimulus-responsive design. In vivo, they exhibit higher targeting of lung cells than free PMB, specifically alveolar and bronchial cells, and effectively treat severe pneumonia in a CRKP (isolated from clinical sample) induced mouse model, markedly improving survival rates. This study proposes a novel, cost-effective method for PMB delivery using PBSi-NPs, potentially broadening the scope of nanomedicine for treating critically ill CRKP-infected patients.

Effect of nystatin on invasion of <i>Serratia grimesii</i> and <i>Serratia proteamaculans</i> bacteria into epithelial cells

BACKGROUND: Bacteria use various endocytic pathways during entering non-phagocytic cells. The involvement of caveolae/lipid rafts in bacterial invasion has been demonstrated for many bacterial pathogens. However, for bacteria of the genus Serratia, the involvement of membrane microdomains in the process of bacterial internalization has been poorly studied. AIM: To evaluate the involvement of caveolae/lipid rafts in the invasion of S. grimesii and S. proteamaculans bacteria into non-phagocytic epithelial Caco-2 and M-HeLa cells using nystatin. MATERIALS AND METHODS: M-HeLa and Caco-2 epithelial cells were incubated with 50 μM nystatin for 1 hour at 37 ºC, after which they were infected with the bacteria S. grimesii strain 30063 and S. proteamaculans strain 94, the multiplicity of infection was 100 bacteria per cell. The number of intracellular bacteria was assessed using gentamicin protection assay. The level of caveolin-1 in cells was visualized using confocal microscopy and Western blotting. The expression of Toll-like receptors genes were measured by real-time RT-PCR. RESULTS: Treatment of epithelial cells with nystatin reduces the internalization of S. grimesii and S. proteamaculans into M-HeLa cells by 30% and does not affect penetration into Caco-2 cells. At the same time, nystatin does not affect the redistribution / the integrity impairment of lipid rafts and does not lead to the cytoskeleton reorganization of eukaryotic cells. The addition of nystatin increases the level of caveolin-1 in M-HeLa cells (caveolin-1 is not expressed in Caco-2), which leads to a change plasma membrane fluidity. Nystatin promotes the secretion of proinflammatory cytokines interleukin-6 and interleukin-8 in both cell lines. Infection of M-HeLa cells pretreated with nystatin with the studied bacteria leads to an increase in the expression of tlr2 and tlr4 genes, but does not exceed the level of their expression in control samples. Therefore, it is impossible to speak unambiguously about the participation of Toll-like receptors in the invasion of Serratia bacteria. CONCLUSIONS: The results obtained suggest that the interaction of bacteria with eukaryotic cells induces the expression of caveolin-1, which leads to a change plasma membrane components mobility. This may be due to the fact that β1-integrin is involved in the invasion of the studied bacteria, which should be stabilized at the plasma membrane upon binding of the ligand due to the formation of a cholesterol- and sphingolipid-rich membrane microenvironment.

Intracellular bactericidal activity and action mechanism of MDP1 antimicrobial peptide against VRSA and MRSA in human endothelial cells.

Staphylococcus aureus is a prominent cause of postoperative infections, often persisting within host cells, leading to chronic infections. Conventional antibiotics struggle to eliminate intracellular S. aureus due to poor cell penetration. Antimicrobial peptides are a new hope for tackling intracellular bacteria. Accordingly, this study examines the antimicrobial peptide MDP1, derived from melittin, for its efficacy against intracellular S. aureus. In this study, the physiochemical properties (Prediction of three-dimensional structure, circular dichroism and helical wheel projection analysis) were investigated. Extracellular antibacterial activity and cytotoxicity of MDP1 were also assessed. The mechanism of interaction of MDP1 with S. aureus was evaluated by molecular dynamic simulation, atomic force and confocal microscopy. Bacterial internalization into an endothelial cell model was confirmed through culture and transmission electron microscopy. The effect of the peptide on intracellular bacteria was investigated by culture and epi-fluorescence microscopy. 3D structural prediction proved the conformation of MDP1 as an α-helix peptide. Helical-wheel projection analysis indicated the proper orientation of hydrophobic amino acid residues for membrane interaction. CD spectroscopy of MDP1 showed that MDP1 in SDS 10 and 30 mM adopted 87 and 91% helical conformation. Atomic force and confocal microscopy assessments as well as molecular dynamics studies revealed the peptide-bacterial membrane interaction. MDP1, at the concentration of 0.32 μg mL-1, demonstrated a fold reduction of 21.7 ± 1.8, 1.7 ± 0.2, and 7.3 ± 0.8 in intracellular bacterial load for ATCC, VRSA, and MRSA, respectively. Molecular dynamics results demonstrate a preferential interaction of MDP1 with POPG/POPE membranes, primarily driven by electrostatic forces and hydrogen bonding. In POPC systems, two out of four MDP1 interacted effectively, while all four MDP1 engaged with POPG/POPE membranes. Gathering all data together, MDP1 is efficacious in the reduction of intracellular VRSA and MRSA proved by culture and epi-fluorescent microscopy although further studies should be performed to increase the intracellular activity of MDP1.

Cutibacterium acnes biofilm formation is influenced by bone microenvironment, implant surfaces and bacterial internalization

BackgroundThe bacterial persistence, responsible for therapeutic failures, can arise from the biofilm formation, which possesses a high tolerance to antibiotics. This threat often occurs when a bone and joint infection is diagnosed after a prosthesis implantation. Understanding the biofilm mechanism is pivotal to enhance prosthesis joint infection (PJI) treatment and prevention. However, little is known on the characteristics of Cutibacterium acnes biofilm formation, whereas this species is frequently involved in prosthesis infections.MethodsIn this study, we compared the biofilm formation of C. acnes PJI-related strains and non-PJI-related strains on plastic support and textured titanium alloy by (i) counting adherent and viable bacteria, (ii) confocal scanning electronic microscopy observations after biofilm matrix labeling and (iii) RT-qPCR experiments.ResultsWe highlighted material- and strain-dependent modifications of C. acnes biofilm. Non-PJI-related strains formed aggregates on both types of support but with different matrix compositions. While the proportion of polysaccharides signal was higher on plastic, the proportions of polysaccharides and proteins signals were more similar on titanium. The changes in biofilm composition for PJI-related strains was less noticeable. For all tested strains, biofilm formation-related genes were more expressed in biofilm formed on plastic that one formed on titanium. Moreover, the impact of C. acnes internalization in osteoblasts prior to biofilm development was also investigated. After internalization, one of the non-PJI-related strains biofilm characteristics were affected: (i) a lower quantity of adhered bacteria (80.3-fold decrease), (ii) an increase of polysaccharides signal in biofilm and (iii) an activation of biofilm gene expressions on textured titanium disk.ConclusionTaken together, these results evidenced the versatility of C. acnes biofilm, depending on the support used, the bone environment and the strain.

Thiolated chitosan nanoparticles encapsulated nisin and selenium: antimicrobial/antibiofilm/anti-attachment/immunomodulatory multi-functional agent

BackgroundThe increase in the resistance of bacterial strains to antibiotics has led to research into the bactericidal potential of non-antibiotic compounds. This study aimed to evaluate in vitro antibacterial/ antibiofilm properties of nisin and selenium encapsulated in thiolated chitosan nanoparticles (N/Se@TCsNPs) against prevalent enteric pathogens including standard isolates of Vibrio (V.) cholerae O1 El Tor ATCC 14,035, Campylobacter (C.) jejuni ATCC 29,428, Salmonella (S.) enterica subsp. enterica ATCC 19,430, Shigella (S.) dysenteriae PTCC 1188, Escherichia (E.) coli O157:H7 ATCC 25,922, Listeria (L.) monocytogenes ATCC 19,115, and Staphylococcus (S.) aureus ATCC 29,733.MethodsThe synthesis and comprehensive analysis of N/Se@TCsNPs have been completed. Antibacterial and antibiofilm capabilities of N/Se@TCsNPs were evaluated through broth microdilution and crystal violet assays. Furthermore, the study included examining the cytotoxic effects on Caco-2 cells and exploring the immunomodulatory effects of N/Se@TCsNPs. This included assessing the levels of both pro-inflammatory (IL-6 and TNFα) and anti-inflammatory (IL-10 and TGFβ) cytokines and determining the gene expression of TLR2 and TLR4.ResultsThe N/Se@TCsNPs showed an average diameter of 136.26 ± 43.17 nm and a zeta potential of 0.27 ± 0.07 mV. FTIR spectroscopy validated the structural features of N/Se@TCsNPs. Scanning electron microscopy (SEM) images confirmed their spherical shape and uniform distribution. Thermogravimetric Analysis (TGA)/Differential Scanning Calorimetry (DSC) tests demonstrated the thermal stability of N/Se@TCsNPs, showing minimal weight loss of 0.03%±0.06 up to 80 °C. The prepared N/Se@TCsNPs showed a thiol content of 512.66 ± 7.33 µmol/g (p < 0.05), an encapsulation efficiency (EE) of 69.83%±0.04 (p ≤ 0.001), and a drug release rate of 74.32%±3.45 at pH = 7.2 (p ≤ 0.004). The synthesized nanostructure demonstrated potent antibacterial activity against various isolates, with effective concentrations ranging from 1.5 ± 0.08 to 25 ± 4.04 mg/mL. The ability of N/Se@TCsNPs to reduce bacterial adhesion and internalization in Caco-2 cells underscored their antibiofilm properties (p ≤ 0.0001). Immunological studies indicated that treatment with N/Se@TCsNPs led to decreased levels of inflammatory cytokines IL-6 (14.33 ± 2.33 pg/mL) and TNFα (25 ± 0.5 pg/mL) (p ≤ 0.0001), alongside increased levels of anti-inflammatory cytokines IL-10 (46.00 ± 0.57 pg/mL) and TGFβ (42.58 ± 2.10 pg/mL) in infected Caco-2 cells (p ≤ 0.0001). Moreover, N/Se@TCsNPs significantly reduced the expression of TLR2 (0.22 ± 0.09) and TLR4 (0.16 ± 0.05) (p < 0.0001).ConclusionIn conclusion, N/Se@TCsNPs exhibited significant antibacterial/antibiofilm/anti-attachment/immunomodulatory effectiveness against selected Gram-positive and Gram-negative enteric pathogens. However, additional ex-vivo and in-vivo investigations are needed to fully assess the performance of nanostructured N/Se@TCsNPs.

Recruitment of Vitronectin by Bacterial Pathogens: A Comprehensive Overview.

The key factor that enables pathogenic bacteria to establish successful infections lies largely in their ability to escape the host's immune response and adhere to host surfaces. Vitronectin (Vn) is a multidomain glycoprotein ubiquitously present in blood and the extracellular matrix of several tissues, where it plays important roles as a regulator of membrane attack complex (MAC) formation and as a mediator of cell adhesion. Vn has emerged as an intriguing target for several microorganisms. Vn binding by bacterial receptors confers protection from lysis resulting from MAC deposition. Furthermore, through its Arg-Gly-Asp (RGD) motif, Vn can bind several host cell integrins. Therefore, Vn recruited to the bacterial cell functions as a molecular bridge between bacteria and host surfaces, where it triggers several host signaling events that could promote bacterial internalization. Each bacterium uses different receptors that recognize specific Vn domains. In this review, we update the current knowledge of Vn receptors of major bacterial pathogens, emphasizing the role they may play in the host upon Vn binding. Focusing on the structural properties of bacterial proteins, we provide details on the residues involved in their interaction with Vn. Furthermore, we discuss the possible involvement of Vn adsorption on biomaterials in promoting bacterial adhesion on abiotic surfaces and infection.

Diazoxon exposure increases susceptibility to infection by Salmonella Typhimurium

ABSTRACT Various exogenous factors, such as microbiological and chemical contamination condition food security. Salmonella Typhimurium (S. Typhimurium) is the cause of salmonellosis. This bacterium utilizes phagocytosis to create bacterial reservoirs. On the other hand, exposure to chemical contaminants, such as pesticides, increases susceptibility to numerous infections. Therefore, this research aims to evaluate the effect of co-exposure to diazoxon and S. Typhimurium on the in vitro infection dynamics. For this purpose, human mononuclear cells were pre-exposed in vitro to diazoxon and then challenged with S. Typhimurium at 1, 8, and 24 h. Bacterial internalization, actin polymerization, and reactive oxygen species (ROS) were analyzed. Obtained data show that mononuclear cells previously exposed to diazoxon exhibit greater internalization of S. Typhimurium. Likewise, greater ROS production and an increase in actin polymerization were observed. Therefore, in the proposed scenario, obtained data suggest that co-exposure to diazoxon and S. Typhimurium increases susceptibility to acquiring an illness.

Role of Rickettsial Outer Membrane Protein A in the Pathogenesis of Rickettsial Diseases

Diseases caused by Rickettsiales are often overlooked, although they pose important public health concerns. The Rickettsiales family comprises a broad range of intracellular bacteria with distinct evolutionary adaptations, making the development of treatment measures to combat infections, such as vaccines or antibiotics, a challenge. Interestingly, the outer membrane protein A (OmpA) was found to exist in the cell surface of most human pathogenic bacteria in the order Rickettsiales. However, knowledge about OmpA in each species and strain is scattered and ambiguous. In this study, we systematically compiled the existing information on OmpA and its relationship with human pathogenic rickettsiae to serve as a reference for future research. A comprehensive literature search was conducted using specific keywords across five databases. According to the literature, OmpA of spotted fever group rickettsia plays a crucial role as an adhesin and invasin that directly interacts with the surface of mammalian host cells to mediate bacterial localization in host cells. The presence of a premature stop codon in the amino acid sequence resulted in the secretion of non-functional OmpA, which is one of the main reasons for rickettsial strains or species to become avirulent. Similarly, OmpA also functions as an important adhesin in the Anaplasma family when it interacts with the sLex and sLex-like glycan of myeloid and endothelial cells, respectively. However, the OmpA of Anaplasma must be co-functional with the other two adhesins to promote bacterial internalization. Interestingly, certain sites in the amino acid residues of Ehrlichia and Orientia OmpA are predicted to be homologous to the binding domain region of Anaplasma OmpA. It is therefore suggested that OmpA is an important adhesin for bacteria to bind to their specific mammalian host cells.

Role of the Putative Histidine Kinase BP1092 in Bordetella pertussis Virulence Regulation and Intracellular Survival.

Bordetella pertussis persists inside host cells, and virulence factors are crucial for intracellular adaptation. The regulation of B. pertussis virulence factor transcription primarily occurs through the modulation of the two-component system (TCS) known as BvgAS. However, additional regulatory systems have emerged as potential contributors to virulence regulation. Here, we investigate the impact of BP1092, a putative TCS histidine kinase that shows increased levels after bacterial internalization by macrophages, on B. pertussis proteome adaptation under nonmodulating (Bvg+) and modulating (Bvg-) conditions. Using mass spectrometry, we compare B. pertussis wild-type (wt), a BP1092-deficient mutant (ΔBP1092), and a ΔBP1092 trans-complemented strain under both conditions. We find an altered abundance of 10 proteins, including five virulence factors. Specifically, under nonmodulating conditions, the mutant strain showed decreased levels of FhaB, FhaS, and Cya compared to the wt. Conversely, under modulating conditions, the mutant strain exhibited reduced levels of BvgA and BvgS compared to those of the wt. Functional assays further revealed that the deletion of BP1092 gene impaired B. pertussis ability to survive within human macrophage THP-1 cells. Taken together, our findings allow us to propose BP1092 as a novel player involved in the intricate regulation of B. pertussis virulence factors and thus in adaptation to the intracellular environment. The data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD041940.

Abstract 122: Fusobacterium nucleatum in saliva of oral cancer patients induces tumor stemness defense phenotype in cancer stem cells

Abstract Background: Oral squamous cell carcinoma (OSCC) is a highly aggressive cancer. Cancer Stem Cells (CSCs) having cell surface markers CD44, ALDH, and EpCAM+ have been identified in OSCC. However, the host pathogen interaction between CSCs and oral microbiome has not yet been studied. A number of studies have recently demonstrated the presence of specific oral bacteria populations and their lipopolysaccharides (LPS) in the tumor microenvironment (1). We previously reported pathogenic bacterial internalization in the CSCs (2). Based on the findings, we have developed an in-vitro model to investigate how oral microbiota may integrate into the tumor microenvironment's CSC population and control its activity. Here, we investigated the host/pathogen interaction between CSCs and FN. Methods: Oral bacteria FN was selectively isolated using FEA agar plates from oral saliva of oral cancer patients (n=10) at stage 3 or 4. The isolated bacteria was co cultured with SCC 25 cells at MOI of 1:50. After co culturing the stemness genes such as SOX 2, OCT 4, Nanog,LPS and ABCG2+ cancer stem cell markers were analysed. ABCG2+ CSCs and ABCG2- cells were immunomagnetically sorted and lysed followed by RNA extraction and RT QPCR was performed to observe if FN has selectively internalised in the CSCs. Also ABCG2+ and ABCG 2- cells were used to perform clonogenic assay. Results: Coculture of FN with SCC 25 cell line induced high expression of stemness genes SOX, OCT 4 and Nanog along with CSC markers ABCG2+. Moreover high expression of LPS was observed indicating the presence of FN in the coculture. Importantly live bacteria was found internalized in ABCG2+ cells but not in ABCG2- cells. High clonogenicity was observed in ABCG2+ CSCs compared to ABCG2- cells. Notably, we found that live bacteria and their LPS, mostly Fusobacterium nucleatum isolated (FN) from clinical subjects, were capable of invading CSCs in the in-vitro setting. Post the host-pathogen interaction; it enabled the activation of a niche modulatory tumor stemness defense (TSD) phenotype in the CSCs. These aggressive CSCs with the TSD phenotype have been found to have a critical role in the progression and relapse of oral cancer. Conclusion: Expression of stemness genes infers the niche defence mechanism of the CSCs of the tumour microenvironment due to infection of FN. Importantly presence of live bacteria in ABCG2+ CSCs indicates that FN selectively infects ABCG2+ CSCs but not ABCG2- cells. High clonogenicity of ABCG2+ CSCs confirms the TSD phenotypic attributes of the CSCs.

Differentiation of keratinocytes or exposure to type 2 cytokines diminishes S. aureus internalization.

Staphylococcus aureus is a leading cause of skin and soft tissue infections. Colonization by this bacterium is increased in individuals with chronic cutaneous diseases such as atopic dermatitis, psoriasis, and bullous pemphigoid. The greater abundance of S. aureus on the skin of subjects with atopic dermatitis in particular has been linked to recurrent cutaneous infections. The primary cell type of the epidermal layer of the skin is the keratinocyte, and it is thought that S. aureus internalized in keratinocytes associates with an increased incidence of skin infections. This study addresses whether keratinocyte differentiation and/or inflammation, two important characteristics altered in cutaneous diseases, influence bacterial internalization. To do this, S. aureus internalization was measured in immortalized and primary keratinocytes that were differentiated using high Ca2+-containing media and/or exposed to cytokines characteristic of atopic dermatitis (IL-4 and IL-13) or psoriasis (IL-17A and IL-22) skin. Our results indicate that S. aureus internalization is uniquely decreased upon keratinocyte differentiation, since this was not observed with another skin-resident bacterium, S. epidermidis. Additionally, treatment with IL-4 + IL-13 diminished bacterial internalization. We interpret this decrease as a mechanism of keratinocyte-based bacterial killing since a similar number of bacterial genomes were detected in cytokine-treated cells, but less viable internalized S. aureus was recovered. Finally, of the receptors reported for S. aureus binding/internalizing into keratinocytes, expression of the α5 component of the α5β1 integrin was in greatest accordance with the number of internalized bacteria in the context of keratinocyte differentiation.IMPORTANCEIndividuals with chronic cutaneous diseases demonstrate heightened susceptibility for severe and recurrent infections from Staphylococcus aureus. What drives this altered susceptibility remains poorly understood. Previous publications have detected S. aureus as deep as the dermal layer of skin in subjects with atopic dermatitis, suggesting that the cutaneous environment of this disease enables deeper bacterial infiltration than occurs in healthy individuals. This observation indicates that S. aureus has greater opportunity to interact with multiple skin cell types in individuals with chronic inflammatory skin diseases. Identifying the characteristics of the skin that influence bacterial internalization, a common method to establish reservoirs and evade the immune response, is critical for our understanding of S. aureus pathogenesis. The significance of this research is the novel identification of epidermal characteristics that influence S. aureus internalization. With this knowledge, methods can be developed to identify patient populations at greater risk for cutaneous infections.

Engagement of α3β1 and α2β1 integrins by hypervirulent Streptococcus agalactiae in invasion of polarized enterocytes.

The gut represents an important site of colonization of the commensal bacterium Streptococcus agalactiae (group B Streptococcus or GBS), which can also behave as a deadly pathogen in neonates and adults. Invasion of the intestinal epithelial barrier is likely a crucial step in the pathogenesis of neonatal infections caused by GBS belonging to clonal complex 17 (CC17). We have previously shown that the prototypical CC17 BM110 strain invades polarized enterocyte-like cells through their lateral surfaces using an endocytic pathway. By analyzing the cellular distribution of putative GBS receptors in human enterocyte-like Caco-2 cells, we find here that the alpha 3 (α3) and alpha 2 (α2) integrin subunits are selectively expressed on lateral enterocyte surfaces at equatorial and parabasal levels along the vertical axis of polarized cells, in an area corresponding to GBS entry sites. The α3β1 and α2β1 integrins were not readily accessible in fully differentiated Caco-2 monolayers but could be exposed to specific antibodies after weakening of intercellular junctions in calcium-free media. Under these conditions, anti-α3β1 and anti-α2β1 antibodies significantly reduced GBS adhesion to and invasion of enterocytes. After endocytosis, α3β1 and α2β1 integrins localized to areas of actin remodeling around GBS containing vacuoles. Taken together, these data indicate that GBS can invade enterocytes by binding to α3β1 and α2β1 integrins on the lateral membrane of polarized enterocytes, resulting in cytoskeletal remodeling and bacterial internalization. Blocking integrins might represent a viable strategy to prevent GBS invasion of gut epithelial tissues.

Thermal‐Cascade Multifunctional Therapeutic Systems for Remotely Controlled Synergistic Treatment of Drug‐Resistant Bacterial Infections

AbstractAntimicrobial therapy remains one of the major global challenges, particularly in the absence of effective treatment strategies for drug‐resistant bacteria. In this study, a comprehensive treatment approach is proposed for drug‐resistant bacterial wound infections based on the development of thermal‐cascade multifunctional therapeutic systems (MTSs), spanning from the design of functional nanoscale materials to macroscopic smart hydrogel. Within the MTSs, functional antibiotic‐loaded hybrid nanoclusters enable targeted therapeutic delivery and synergistic mild hyperthermia‐antibiotic treatment, strongly suppressing drug‐resistant bacteria while demonstrating excellent biocompatibility with normal cells and tissues. Furthermore, near‐infrared irradiation can trigger the photothermal effects of hybrid nanoclusters to induce gelation of thermal‐sensitive hydrogel, forming MTSs to serve as highly adaptable, drug‐enriched protective dressings for infected wounds. Both in vitro and in vivo results substantiate that MTSs enhance the bioavailability of therapeutic agents (including bacterial internalization and tissue penetration), exert synergistic effects to completely eradicate drug‐resistant bacteria, promoted wound healing and revascularization, and demonstrate excellent biocompatibility. This work offers an innovative demonstration to address drug‐resistant bacterial infections through the advancement of sophisticated material systems.

Anti-OmpA antibodies as potential inhibitors of Acinetobacter baumannii biofilm formation, adherence to, and proliferation in A549 human alveolar epithelial cells

Outer membrane protein A (OmpA) is a critical virulence factor in Acinetobacter baumannii, influencing adhesion, biofilm formation, host immune response, and host cell apoptosis. We investigated the invasion of A549 alveolar epithelial cells by A. baumannii and examined how anti-OmpA antibodies impact these interactions. OmpA was expressed and purified, inducing anti-OmpA antibodies in BALB/c mice. The potential toxicity of OmpA was evaluated in mice by analyzing histology from six organs. A549 cells were exposed to A. baumannii strains 19606 and a clinical isolate. Using cell culture and light microscopy, we scrutinized the effects of anti-OmpA sera on serum resistance, adherence, internalization, and proliferation of A. baumannii in A549 cells. The viability of A549 cells was assessed upon exposure to live A. baumannii and anti-OmpA sera. OmpA-induced antibody demonstrated potent bactericidal effects on both strains of A. baumannii. Both strains formed biofilms, which were reduced by anti-OmpA serum, along with decreased bacterial adherence, internalization, and proliferation in A549 cells. Anti-OmpA serum improved the survival of A549 cells post-infection. Pre-treatment with cytochalasin D hindered bacterial internalization, highlighting the role of actin polymerization in invasion. Microscopic examination revealed varied interactions encompassing adherence, apoptosis, membrane alterations, vacuolization, and damage. A549 cells treated with anti-OmpA serum exhibited improved structures and reduced damage. The findings indicate that A. baumannii can adhere to and proliferate within epithelial cells with OmpA playing a pivotal role in these interactions, and the complex nature of these interactions shapes the intricate course of A. baumannii infection in host cells.

Platelet Factor 4 Enhances Antimicrobial Function of the Endothelium and Improves Outcome in a Murine Model of Sepsis

Introduction: In this study, we investigated mechanism(s) by which the platelet-specific chemokine platelet factor 4 (PF4) interacts with bacteria to enhance host-defense function and improve outcome in sepsis. Sepsis, a life-threatening dysregulated response to infection, is the leading cause of mortality worldwide for which there are no targeted treatments. Upon sensing pathogens, platelets release high concentrations of PF4, a positively charged chemokine with high affinity for polyanions including polymers in the bacterial cell wall, the endothelial glycocalyx and cell-free DNA. We previously found that PF4 improves outcomes in the murine lipopolysaccharide endotoxemia and enhances in vitro bacterial capture by neutrophil extracellular traps. We now show that PF4-coating of bacteria reduces coagulopathy, limits bacterial dissemination, and improves survival in a murine model of polymicrobial sepsis, in part through the novel mechanism of enhancing the antimicrobial function of endothelial cells (ECs). Methods: Effects of PF4 on bacterial internalization by ECs was assessed by incubating heat-inactivated Staphylococcus (S) aureus conjugated to pHrodo, a dye that only fluoresces following cellular internalization, with PF4 (0-100mg/mL) and then expose those complexes to human umbilical vein endothelial cells (HUVECs) ± tumor necrosis factor a (TNFa) to simulate inflammation. Internalized S aureus fluorescent signal was visualized 16hr post-exposure. HUVECs were stained for von Willebrand factor (VWF) release to indicate bacteria-induced endothelial activation. Effects of PF4 on EC killing of bacteria was studied by exposing HUVECs to live Escherichia (E) coli pre-treated with PF4 (0-25mg/mL) for 1hr. ECs were washed to remove excess unbound bacteria, and cell lysates were plated on agar plates for 24hr to count colony forming units (CFUs). To investigate the effects of PF4-bacteria interactions on sepsis survival outcomes, contents from the cecum (cecal slurry, CS) of C57BL6 donor mice (100mg/mL) were pre-treated with PF4 (100mg) for 20min and injected intravenously (IV) into wildtype (WT) or PF4 -/-recipient mice to induce immediate bacteremia. Animals were evaluated for sepsis severity for up to 72hr, and survival analysis was performed using the Mantel-Cox test. A separate cohort of WT mice was given IV CS (400mg/mL) pre-incubated with PF4 (8mg). Blood was drawn at 3 to 24hr post-CS infusion and subjected to complete blood count. Thrombin anti-thrombin (TAT) and VWF levels were measured by ELISAs in plasma as indicators of intravascular coagulopathy and endothelial activation. To assess bacterial load, blood and liver and spleen homogenates were plated on agar plates to count for CFUs. Results: PF4 coating of bacteria enhanced bacterial uptake by HUVECs, peaking at 20-50 µg/mL of PF4. TNFa-stimulated HUVECs more readily internalized PF4-coated bacteria at 1-20 µg/mL of PF4. Although PF4 promoted S aureus uptake, it limited bacteria-induced VWF secretion by HUVECs. PF4 also enhanced EC killing of E coli upon bacterial internalization. In the CS model of murine polymicrobial sepsis, PF4 prevented CS-induced mortality in both WT and PF4 -/- mice ( Fig. 1). PF4-pretreatment of CS also reduced leukopenia and thrombocytopenia 3-6hr post-CS challenge in WT mice. Mice given PF4-treated CS exhibited lower TAT and VWF levels as compared to untreated-CS animals. Lastly, PF4 reduced bacterial load in blood, liver, and spleen 24hr post-CS inoculation ( Fig. 2). Conclusion: PF4 enhances bacterial uptake and killing by the endothelium in vitro, promotes bacterial clearance while preventing bacteria-induced coagulopathy and endothelial dysfunction leading to improved survival in the murine CS model of polymicrobial sepsis. These studies suggest that PF4 binding to bacteria may be an important host defense mechanism. Further studies are underway to define the contribution of EC antimicrobial activity to the observed outcomes. The ability of PF4-based therapeutics to enhance clearance of microbes merits further translational studies in sepsis.