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The Gram-positive bacterium, Filifactor alocis is an oral pathogen, and approximately 50% of known strains encode a recently identified repeat-in-toxin (RTX) protein, FtxA. By assessing a longitudinal Ghanaian study population of adolescents (10-19 years of age; mean age 13.2 years), we recently discovered a possible correlation between deep periodontal pockets measured at the two-year follow-up, presence of the ftxA gene, and a high quantity of F. alocis. To further understand the contribution of F. alocis and FtxA in periodontal disease, we used qPCR in the present study to assess the carriage loads of F. alocis and the prevalence of its ftxA gene in subgingival plaque specimens, sampled at baseline from the Ghanaian cohort (n=500). Comparing these results with the recorded clinical attachment loss (CAL) longitudinal progression data from the two-year follow up, we concluded that carriers of ftxA-positive F. alocis typically exhibited higher loads of the bacterium. Moreover, high carriage loads of F. alocis and concomitant presence of the ftxA gene were two factors that were both associated with an enhanced prevalence of CAL progression. Interestingly, CAL progression appeared to be further promoted upon the simultaneous presence of F. alocis and the non-JP2 genotype of Aggregatibacter actinomycetemcomitans. Taken together, our present findings are consistent with the notion that F. alocis and its ftxA gene promotes CAL during periodontal disease. [ABSTRACT FROM AUTHOR]

The Gram-positive bacterium, Filifactor alocis is an oral pathogen, and approximately 50% of known strains encode a recently identified repeat-in-toxin (RTX) protein, FtxA. By assessing a longitudinal Ghanaian study population of adolescents (10-19 years of age; mean age 13.2 years), we recently discovered a possible correlation between deep periodontal pockets measured at the two-year follow-up, presence of the ftxA gene, and a high quantity of F. alocis. To further understand the contribution of F. alocis and FtxA in periodontal disease, we used qPCR in the present study to assess the carriage loads of F. alocis and the prevalence of its ftxA gene in subgingival plaque specimens, sampled at baseline from the Ghanaian cohort (n=500). Comparing these results with the recorded clinical attachment loss (CAL) longitudinal progression data from the two-year follow up, we concluded that carriers of ftxA-positive F. alocis typically exhibited higher loads of the bacterium. Moreover, high carriage loads of F. alocis and concomitant presence of the ftxA gene were two factors that were both associated with an enhanced prevalence of CAL progression. Interestingly, CAL progression appeared to be further promoted upon the simultaneous presence of F. alocis and the non-JP2 genotype of Aggregatibacter actinomycetemcomitans. Taken together, our present findings are consistent with the notion that F. alocis and its ftxA gene promotes CAL during periodontal disease.

The acylated pore-forming R epeats in T o X in (RTX) cytolysins α-hemolysin (HlyA) and adenylate cyclase toxin (CyaA) preferentially bind to β 2 integrins of myeloid leukocytes but can also promiscuously bind and permeabilize cells lacking the β 2 integrins. We constructed a HlyA 1 – 563 /CyaA 860 – 1706 chimera that was acylated either by the toxin-activating acyltransferase CyaC, using sixteen carbon-long (C16) acyls, or by the HlyC acyltransferase using fourteen carbon-long (C14) acyls. Cytolysin assays with the C16- or C14-acylated HlyA/CyaA chimeric toxin revealed that the RTX domain of CyaA can functionally replace the RTX domain of HlyA only if it is modified by C16-acyls on the Lys983 residue of CyaA. The C16-monoacylated HlyA/CyaA chimera was as pore-forming and cytolytic as native HlyA, whereas the C14-acylated chimera exhibited very low pore-forming activity. Hence, the capacity of the RTX domain of CyaA to support the insertion of the N-terminal pore-forming domain into the target cell membrane, and promote formation of toxin pores, strictly depends on the modification of the Lys983 residue by an acyl chain of adapted length. [Display omitted] • A palmitoylated HlyA/CyaA chimera is as cytolytic as myristoylated HlyA toxin. • Myristoylation (C14-acylation) fails to activate the HlyA/CyaA chimera. • Palmitoylated (C16-acylated) HlyA/CyaA chimera forms HlyA-like pores. • This HlyA/CyaA chimera binds the β 2 integrin CR3 (CD11b/CD18). [ABSTRACT FROM AUTHOR]

The Gram-negative bacterium Kingella kingae is part of the commensal oropharyngeal flora of young children. As detection methods have improved, K. kingae has been increasingly recognized as an emerging invasive pathogen that frequently causes skeletal system infections, bacteremia, and severe forms of infective endocarditis. K. kingae secretes an RtxA cytotoxin, which is involved in the development of clinical infection and belongs to an ever-growing family of cytolytic RTX (Repeats in ToXin) toxins secreted by Gram-negative pathogens. All RTX cytolysins share several characteristic structural features: (i) a hydrophobic pore-forming domain in the N-terminal part of the molecule; (ii) an acylated segment where the activation of the inactive protoxin to the toxin occurs by a co-expressed toxin-activating acyltransferase; (iii) a typical calcium-binding RTX domain in the C-terminal portion of the molecule with the characteristic glycine- and aspartate-rich nonapeptide repeats; and (iv) a C-proximal secretion signal recognized by the type I secretion system. RTX toxins, including RtxA from K. kingae, have been shown to act as highly efficient 'contact weapons' that penetrate and permeabilize host cell membranes and thus contribute to the pathogenesis of bacterial infections. RtxA was discovered relatively recently and the knowledge of its biological role remains limited. This review describes the structure and function of RtxA in the context of the most studied RTX toxins, the knowledge of which may contribute to a better understanding of the action of RtxA in the pathogenesis of K. kingae infections. [ABSTRACT FROM AUTHOR]

Post-translational acylation of lysine side chains is a common mechanism of protein regulation. Modification by long-chain fatty acyl groups is an understudied form of lysine acylation that has gained increasing attention recently due to the characterization of enzymes that catalyze the addition and removal this modification. In this review we summarize what has been learned about lysine fatty acylation in the approximately 30 years since its initial discovery. We report on what is known about the enzymes that regulate lysine fatty acylation and their physiological functions, including tumorigenesis and bacterial pathogenesis. We also cover the effect of lysine fatty acylation on reported substrates. Generally, lysine fatty acylation increases the affinity of proteins for specific cellular membranes, but the physiological outcome depends greatly on the molecular context. Finally, we will go over the experimental tools that have been used to study lysine fatty acylation. While much has been learned about lysine fatty acylation since its initial discovery, the full scope of its biological function has yet to be realized.

Moraxella bovoculi is the bacterium most often cultured from ocular lesions of cattle with infectious bovine keratoconjunctivitis, also known as bovine pinkeye. Some strains of M. bovoculi contain operons encoding for a repeats-in-toxin (RTX) toxin, which is a known virulence factor of multiple veterinary pathogens. We explored the utility of MALDI-TOF MS and biomarker detection models to classify the presence or absence of an RTX phenotype in M. bovoculi. Ninety strains that had undergone whole genome sequencing were classified by the presence or absence of complete RTX operons and confirmed with a visual assessment of hemolysis on blood agar. Strains were grown on Tryptic Soy Agar (TSA) with 5% sheep blood, TSA with 5% bovine blood that was supplemented with 10% fetal bovine serum, 10 mmol/LCaCl2, or both. The formulations were designed to determine the influence of growth media on toxin production or activity, as calcium ions are required for toxin secretion and activity. Mass spectra were obtained for strains grown on each agar formulation and biomarker models were developed using ClinProTools 3.0 software. The most accurate model was developed using spectra from strains grown on TSA with 5% bovine blood and supplemented with CaCl2, which had a sensitivity and specificity of 93.3% and 73.3%, respectively, regarding RTX phenotype classification. The same biomarker model algorithm developed from strains grown on TSA with 5% sheep blood had a substantially lower sensitivity and specificity of 68.0% and 52.0%, respectively. Our results indicate that MALDI-TOF MS biomarker models can accurately classify strains of M. bovoculi regarding the presence or absence of RTX toxin operons and that agar media modifications improve the accuracy of these models.

Moraxella bovoculi is the bacterium most often cultured from ocular lesions of cattle with infectious bovine keratoconjunctivitis, also known as bovine pinkeye. Some strains of M. bovoculi contain operons encoding for a repeats-in-toxin (RTX) toxin, which is a known virulence factor of multiple veterinary pathogens. We explored the utility of MALDI-TOF MS and biomarker detection models to classify the presence or absence of an RTX phenotype in M. bovoculi. Ninety strains that had undergone whole genome sequencing were classified by the presence or absence of complete RTX operons and confirmed with a visual assessment of hemolysis on blood agar. Strains were grown on Tryptic Soy Agar (TSA) with 5% sheep blood, TSA with 5% bovine blood that was supplemented with 10% fetal bovine serum, 10 mmol/LCaCl2, or both. The formulations were designed to determine the influence of growth media on toxin production or activity, as calcium ions are required for toxin secretion and activity. Mass spectra were obtained for strains grown on each agar formulation and biomarker models were developed using ClinProTools 3.0 software. The most accurate model was developed using spectra from strains grown on TSA with 5% bovine blood and supplemented with CaCl2, which had a sensitivity and specificity of 93.3% and 73.3%, respectively, regarding RTX phenotype classification. The same biomarker model algorithm developed from strains grown on TSA with 5% sheep blood had a substantially lower sensitivity and specificity of 68.0% and 52.0%, respectively. Our results indicate that MALDI-TOF MS biomarker models can accurately classify strains of M. bovoculi regarding the presence or absence of RTX toxin operons and that agar media modifications improve the accuracy of these models. [ABSTRACT FROM AUTHOR]

The Gram-negative bacterium Kingella kingae is part of the commensal oropharyngeal flora of young children. As detection methods have improved, K. kingae has been increasingly recognized as an emerging invasive pathogen that frequently causes skeletal system infections, bacteremia, and severe forms of infective endocarditis. K. kingae secretes an RtxA cytotoxin, which is involved in the development of clinical infection and belongs to an ever-growing family of cytolytic RTX (Repeats in ToXin) toxins secreted by Gram-negative pathogens. All RTX cytolysins share several characteristic structural features: (i) a hydrophobic pore-forming domain in the N-terminal part of the molecule; (ii) an acylated segment where the activation of the inactive protoxin to the toxin occurs by a co-expressed toxin-activating acyltransferase; (iii) a typical calcium-binding RTX domain in the C-terminal portion of the molecule with the characteristic glycine- and aspartate-rich nonapeptide repeats; and (iv) a C-proximal secretion signal recognized by the type I secretion system. RTX toxins, including RtxA from K. kingae, have been shown to act as highly efficient ‘contact weapons’ that penetrate and permeabilize host cell membranes and thus contribute to the pathogenesis of bacterial infections. RtxA was discovered relatively recently and the knowledge of its biological role remains limited. This review describes the structure and function of RtxA in the context of the most studied RTX toxins, the knowledge of which may contribute to a better understanding of the action of RtxA in the pathogenesis of K. kingae infections.

ABSTRACT The Escherichia coli hemolysin (HlyA) is a pore-forming exotoxin associated with severe complications of human urinary tract infections. HlyA is the prototype of the repeats-in-toxin (RTX) family, which includes LtxA from Aggregatibacter actinomycetemcomitans, a periodontal pathogen. The existence and requirement for a host cell receptor for these toxins are controversial. We performed an unbiased forward genetic selection in a mutant library of human monocytic cells, U-937, for host factors involved in HlyA cytotoxicity. The top candidate was the β2 integrin β subunit. Δβ2 cell lines are approximately 100-fold more resistant than wild-type U-937 cells to HlyA, but remain sensitive to HlyA at high concentrations. Similarly, Δβ2 cells are more resistant than wild-type U-937 cells to LtxA, as Δβ2 cells remain LtxA resistant even at >1,000-fold-higher concentrations of the toxin. Loss of any single β2 integrin α subunit, or even all four α subunits together, does not confer resistance to HlyA. HlyA and LtxA bind to the β2 subunit, but not to αL, αM, or αX in far-Western blots. Genetic complementation of Δβ2 cells with either β2 or β2 with a cytoplasmic tail deletion restores HlyA and LtxA sensitivity, suggesting that β2 integrin signaling is not required for cytotoxicity. Finally, β2 mutations do not alter sensitivity to unrelated pore-forming toxins, as wild-type or Δβ2 cells are equally sensitive to Staphylococcus aureus α-toxin and Proteus mirabilis HpmA. Our studies show two RTX toxins use the β2 integrin β subunit alone to facilitate cytotoxicity, but downstream integrin signaling is dispensable. IMPORTANCE Urinary tract infections are one of the most common bacterial infections worldwide. Uropathogenic Escherichia coli strains are responsible for more than 80% of community-acquired urinary tract infections. Although we have known for nearly a century that severe infections stemming from urinary tract infections, including kidney or bloodstream infections are associated with expression of a toxin, hemolysin, from uropathogenic Escherichia coli, how hemolysin functions to enhance virulence is unknown. Our research defines the interaction of hemolysin with the β2 integrin, a human white cell adhesion molecule, as a potential therapeutic target during urinary tract infections. The E. coli hemolysin is the prototype for a toxin family (RTX family) produced by a wide array of human and animal pathogens. Our work extends to the identification and characterization of the receptor for an additional member of the RTX family, suggesting that this interaction may be broadly conserved throughout the RTX toxin family.

Abstract Background Catechins, polyphenols derived from tea leaves, have been shown to have antibacterial properties, through direct killing of bacteria as well as through inhibition of bacterial toxin activity. In particular, certain catechins have been shown to have bactericidal effects on the oral bacterium, Aggregatibacter actinomycetemcomitans , as well as the ability to inhibit a key virulence factor of this organism, leukotoxin (LtxA). The mechanism of catechin-mediated inhibition of LtxA has not been shown. Methods In this work, we studied the ability of six catechins to inhibit LtxA-mediated cytotoxicity in human white blood cells, using Trypan blue staining, and investigated the mechanism of action using a combination of techniques, including fluorescence and circular dichroism spectroscopy, confocal microscopy, and surface plasmon resonance. Results We found that all the catechins except (−)-catechin inhibited the activity of this protein, with the galloylated catechins having the strongest effect. Pre-incubation of the toxin with the catechins increased the inhibitory action, indicating that the catechins act on the protein, rather than the cell. The secondary structure of LtxA was dramatically altered in the presence of catechin, which resulted in an inhibition of toxin binding to cholesterol, an important initial step in the cytotoxic mechanism of the toxin. Conclusions These results demonstrate that the catechins inhibit LtxA activity by altering its structure to prevent interaction with specific molecules present on the host cell surface. General significance Galloylated catechins modify protein toxin structure, inhibiting the toxin from binding to the requisite molecules on the host cell surface. Graphical abstract Unlabelled Image Highlights • Galloylated catechins alter the structure of A. actinomycetemcomitans leukotoxin. • Galloylated catechins prevent leukotoxin from binding to cholesterol. • Leukotoxin activity is thus greatly inhibited by galloylated catechins. [ABSTRACT FROM AUTHOR]

Leukotoxin (LtxA), from oral pathogen Aggregatibacter actinomycetemcomitans, is a secreted membrane-damaging protein. LtxA is internalized by β2 integrin LFA-1 (CD11a/CD18)-expressing leukocytes and ultimately causes cell death; however, toxin localization in the host cell is poorly understood and these studies fill this void. We investigated LtxA trafficking using multi-fluor confocal imaging, flow cytometry and Rab5a knockdown in human T lymphocyte Jurkat cells. Planar lipid bilayers were used to characterize LtxA pore-forming activity at different pHs. Our results demonstrate that the LtxA/LFA-1 complex gains access to the cytosol of Jurkat cells without evidence of plasma membrane damage, utilizing dynamin-dependent and presumably clathrin-independent mechanisms. Upon internalization, LtxA follows the LFA-1 endocytic trafficking pathways, as identified by co-localization experiments with endosomal and lysosomal markers (Rab5, Rab11A, Rab7, and Lamp1) and CD11a. Knockdown of Rab5a resulted in the loss of susceptibility of Jurkat cells to LtxA cytotoxicity, suggesting that late events of LtxA endocytic trafficking are required for toxicity. Toxin trafficking via the degradative endocytic pathway may culminate in the delivery of the protein to lysosomes or its accumulation in Rab11A-dependent recycling endosomes. The ability of LtxA to form pores at acidic pH may result in permeabilization of the endosomal and lysosomal membranes.

T helper type 17 (Th17) cells are a subset of pro-inflammatory T helper cells that mediate host defense and pathological inflammation. We have previously reported that host dendritic cells (DCs) infected with Vibrio vulnificus induce Th17 responses through the production of several pro-inflammatory cytokines, including interleukin (IL)-1β and IL-6. V. vulnificus produces RTX toxin (RtxA), an important virulence factor that determines successful pathophysiology. In this study, we investigated the involvement of RtxA from V. vulnificus in Th17 cell induction through the activation and maturation of DCs. The increased expression of the DC surface marker CD40 caused by V. vulnificus wild-type infection was reduced by rtxA gene mutation in V. vulnificus. The mRNA and protein levels of Th17 polarization-related cytokines also decreased in V. vulnificus rtxA mutant-infected DCs. In addition, the co-culture of Th cells and DCs infected with rtxA mutant V. vulnificus resulted in reduction in DC-mediated Th17 responses. Th17 cell responses in the small intestinal lamina propria decreased in mice inoculated with V. vulnificus rtxA mutant as compared to those inoculated with the wild-type strain. These decreases in DC maturation, Th17-polarizing cytokine secretion, and Th17 responses attributed to rtxA mutation were restored following infection with the rtxA revertant strain. Furthermore, the mutation in the hlyU gene encoding the activator of rtxA1 gene reproduced the results observed with rtxA mutation. Taken together, V. vulnificus, by means of RtxA, induces inflammatory Th17 responses, which may be associated with adaptive responses of the host against V. vulnificus infection.

A leukotoxin (LtxA) that is produced by Aggregatibacter actinomycetemcomitans (Aa) is an important virulence determinant in an aggressive form of periodontitis in adolescents. Understanding the function of this protein at the molecular level is critical to elucidating its role in the disease process. To accomplish genetic analysis of the protein structure and relating these observations to toxin function, we have developed an E. coli expression system for the generation and rapid purification of LtxA. Cloning the structural toxin gene, ltxA, from Aa strain JP2 under control of T7 promoter-1 of pCDFDuet-1 vector resulted in expression of a 114 KDa protein which could be easily purified by the presence of a carboxy-terminal engineered double hexahistidine (double–His 6 ) tag and was immunologically reactive with an anti-LtxA monoclonal antibody, but was not cytotoxic. Cloning a second gene, ltxC, an acyltransferase gene, into the vector under control of T7 promoter-2, resulted in expression of the biologically active LtxA. The toxin was extracted from E. coli inclusion bodies, purified by immobilized metal affinity chromatography, and refolded by dialysis. When compared by circular dichroism (CD) spectroscopy analysis, acylated recombinant LtxA has a secondary structure consistent with wt LtxA, while variations in α-helical structure of nonacylated LtxA were observed. No modifications in α-helix were found upon the toxin's binding with liposome-incorporated cholesterol. Our results suggest that pure, biologically active recombinant LtxA can be isolated by a one-step affinity chromatography from E. coli . The toxic and structural properties of the recombinant LtxA are similar to its wt counterpart. [ABSTRACT FROM AUTHOR]

T helper type 17 (Th17) cells are a subset of pro-inflammatory T helper cells that mediate host defense and pathological inflammation. We have previously reported that host dendritic cells (DCs) infected with Vibrio vulnificus induce Th17 responses through the production of several pro-inflammatory cytokines, including interleukin (IL)-1β and IL-6. V. vulnificus produces RTX toxin (RtxA), an important virulence factor that determines successful pathophysiology. In this study, we investigated the involvement of RtxA from V. vulnificus in Th17 cell induction through the activation and maturation of DCs. The increased expression of the DC surface marker CD40 caused by V. vulnificus wild-type infection was reduced by rtxA gene mutation in V. vulnificus. The mRNA and protein levels of Th17 polarization-related cytokines also decreased in V. vulnificus rtxA mutant-infected DCs. In addition, the co-culture of Th cells and DCs infected with rtxA mutant V. vulnificus resulted in reduction in DC-mediated Th17 responses. Th17 cell responses in the small intestinal lamina propria decreased in mice inoculated with V. vulnificus rtxA mutant as compared to those inoculated with the wild-type strain. These decreases in DC maturation, Th17-polarizing cytokine secretion, and Th17 responses attributed to rtxA mutation were restored following infection with the rtxA revertant strain. Furthermore, the mutation in the hlyU gene encoding the activator of rtxA1 gene reproduced the results observed with rtxA mutation. Taken together, V. vulnificus , by means of RtxA, induces inflammatory Th17 responses, which may be associated with adaptive responses of the host against V. vulnificus infection. [ABSTRACT FROM AUTHOR]

Type I secretion systems are widespread in Gram-negative bacteria and mediate the one-step translocation of a large variety of proteins serving for diverse purposes, including nutrient acquisition or bacterial virulence. Common to most substrates of type I secretion systems is the presence of a C-terminal secretion sequence that is not cleaved during or after translocation. Furthermore, these protein secretion nanomachineries are always composed of an ABC transporter, a membrane fusion protein, both located in the inner bacterial membrane, and a protein of the outer membrane. These three membrane proteins transiently form a 'tunnel channel' across the periplasmic space in the presence of the substrate. Here we summarize the recent findings with respect to structure, function and application of type I secretion systems. [ABSTRACT FROM AUTHOR]

RTX proteins are a large family of polypeptides of mainly Gram-negative origin that are secreted into the extracellular medium by a type I secretion system featuring a non-cleavable C-terminal secretion signal, which is preceded by a variable number of nine-residue tandem repeats. The three-dimensional structure forms a parallel β-roll, where β-strands of two parallel sheets are connected by calcium-binding linkers in such a way that a right-handed spiral is built. The Ca2+ ions are an integral part of the structure, which cannot form without them. The structural determinants of this unique architecture will be reviewed with its conservations and variations together with the implication for secretion and folding of these proteins. The general purpose of the RTX domains appears to act as an internal chaperone that keeps the polypeptide unfolded in the calcium-deprived cytosol and triggers folding in the calcium-rich extracellular medium. A rather recent addition to the structural biology of the RTX toxin is a variant occurring in a large RTX adhesin, where this non-canonical β-roll binds to ice and diatoms.

Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ≈400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called "two-step model" and the recently-proposed "toroidal pore model", will be considered. [ABSTRACT FROM AUTHOR]

The acylated Repeats in ToXins (RTX) leukotoxins, the adenylate cyclase toxin (CyaA) or α-hemolysin (HlyA), bind β 2 integrins of leukocytes but also penetrate cells lacking these receptors. We show that the indoles of conserved tryptophans in the acylated segments, W876 of CyaA and W579 of HlyA, are crucial for β 2 integrin-independent membrane penetration. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding, or the activities of CyaA W876L/F/Y variants on cells expressing high amounts of the β 2 integrin CR3. However, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking β 2 integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (T m ) of CyaA by 4 to 8 °C but locally enhanced the accessibility to deuteration of the hydrophobic segment and of the interface of the two acylated loops. W876Q substitution (showing no increase in T m ), or combination of W876F with a cavity-filling V822M substitution (this combination decreasing the T m closer to that of CyaA), yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA on erythrocytes was also selectively impaired when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. Hence, the bulky indoles of residues W876 of CyaA, or W579 of HlyA, rule the local positioning of the acylated loops and enable a membrane-penetrating conformation in the absence of RTX toxin docking onto the cell membrane by β 2 integrins., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Aggregatibacter actinomycetemcomitans is an oral pathogen and etiologic agent of localized aggressive periodontitis. The bacterium is also a cardiovascular pathogen causing infective endocarditis. A. actinomycetemcomitans produces leukotoxin (LtxA), an important virulence factor that targets white blood cells (WBCs) and plays a role in immune evasion during disease. The functional receptor for LtxA on WBCs is leukocyte function antigen-1 (LFA-1), a b-2 integrin that is modified with N-linked carbohydrates. Interaction between toxin and receptor leads to cell death. We recently discovered that LtxA can also lyse red blood cells (RBCs) and hemolysis may be important for pathogenesis of A. actinomycetemcomitans. In this study, we further investigated how LtxA might recognize and lyse RBCs. We found that, in contrast to a related toxin, E. coli a-hemolysin, LtxA does not recognize glycophorin on RBCs. However, gangliosides were able to completely block LtxA-mediated hemolysis. Furthermore, LtxA did not show a preference for any individual ganglioside. LtxA also bound to ganglioside-rich C6 rat glioma cells, but did not kill them. Interaction between LtxA and C6 cells could be blocked by gangliosides with no apparent specificity. Gangliosides were only partially effective at preventing LtxA-mediated cytotoxicity of WBCs, and the effect was only observed when a high ratio of ganglioside:LtxA was used over a short incubation period. Based on the results presented here, we suggest that because of the similarity between N-linked sugars on LFA-1 and the structures of gangliosides, LtxA may have acquired the ability to lyse RBCs.

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, has been associated with localized aggressive periodontitis (LAP). In particular, highly leukotoxic strains of A. actinomycetemcomitans have been more closely associated with this disease, suggesting that LtxA is a key virulence factor for A. actinomycetemcomitans. LtxA is secreted across both the inner and outer membranes via the Type I secretion system, but has also been found to be enriched within outer membrane vesicles (OMVs), derived from the bacterial outer membrane. We have characterized the association of LtxA with OMVs produced by the highly leukotoxic strain, JP2, and investigated the interaction of these OMVs with host cells to understand how LtxA is delivered to host cells in this OMV-associated form. Our results demonstrated that a significant fraction of the secreted LtxA exists in an OMV-associated form. Furthermore, we have discovered that in this OMV-associated form, the toxin is trafficked to host cells by a cholesterol- and receptor-independent mechanism in contrast to the mechanism by which free LtxA is delivered. Because OMV-associated toxin is trafficked to host cells in an entirely different manner than free toxin, this study highlights the importance of studying both free and OMV-associated forms of LtxA to understand A. actinomycetemcomitans virulence.

Hemolysin from uropathogenic Escherichia coli (UPEC) is a hemolytic and cytotoxic protein active against a broad range of species and cell types. Expression of hemolysin correlates with severity of infection, as up to 78% of UPEC isolates from pyelonephritis cases express hemolysin. Despite decades of research on hemolysin activity, the mechanism of intoxication and the function of hemolysin in UPEC infection remain elusive. Early in vitro research established the role of hemolysin as a lytic protein at high doses. It is hypothesized that hemolysin is secreted at sublytic doses in vivo and recent research has focused on understanding the more subtle effects of hemolysin both i n vitro and in elegant infection models in vivo , including inoculation by micropuncture of individual kidney nephrons. As the field continues to evolve, comparisons of hemolysin function in isolates from a range of UTI infections will be important for delineating the role of this toxin. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale. [ABSTRACT FROM AUTHOR]

Studies have suggested that Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) kills human lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18)-bearing immune cells through a lysosomal-mediated mechanism. Lysosomes are membrane-bound cellular organelles that contain an array of acid hydrolases that are capable of breaking down biomolecules. The lysosomal membrane bilayer confines the pH-sensitive enzymes within an optimal acidic (pH 4.8) environment thereby protecting the slightly basic cytosol (pH 6.8-7.5). In the current study, we have probed the effect of LtxA-induced cytolysis on lysosomal integrity in two different K562 erythroleukemia cell lines. K562-puro/LFA-1 cells were stably transfected with CD11a and CD18 cDNA to express LFA-1 on the cell surface while K562-puro, which does not express LFA-1, served as a control. Following treatment with 100 ng ml(-1) LtxA cells were analyzed by live cell imaging in conjunction with time-lapse confocal microscopy and by flow cytometry. Using a pH-sensitive indicator (pHrodo(®)) we demonstrated that the toxin causes a decrease in the intracellular pH in K562-puro/LFA-1 cells that is noticeable within the first 15 min of treatment. This process correlated with the disappearance of lysosomes in the cytosol as determined by both acridine orange and LysoTracker(®) Red DND-99 staining. These changes were not observed in K562-puro cells or when heat inactivated toxin was added to K562-puro/LFA-1. Our results suggest that LtxA induces lysosomal damage, cytosol acidification, which is followed by cell death in K562-puro/LFA-1 cells. [ABSTRACT FROM AUTHOR]

Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ≈400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called “two-step model” and the recently-proposed “toroidal pore model”, will be considered.

Uropathogenic strains of Escherichia coli deliver the toxin alpha-hemolysin (HlyA) to optimize the host environment for the spread of infection. It was reported that at high concentrations, the toxin forms pores in eukaryotic membranes, leading to cell lysis, while lower concentrations have appeared to interfere with host–cell-signaling pathways causing cell death by apoptosis. Nevertheless, what is not clear is how often HlyA reaches levels that are high enough to lyse host target cells during the course of an infection. In the present investigation, we demonstrate that a low toxin concentration induces the suicidal death of erythrocytes (eryptosis), the major cell type present in blood. Eryptosis is triggered both by an increment in intracellular calcium and by ceramide. Since we have previously demonstrated that a low concentration of HlyA induces an increase in intraerythrocyte calcium, in the present experiments we have shown that this ion activates calpains, which hydrolyze skeleton proteins such as spectrin, ankyrin, protein 4.1 and the electrophoretic Band-3 species, thus resulting in morphologic changes in the erythrocytes. We furthermore observed that a low toxin concentration induced the activation of endogenous sphingomyelinases that in turn increased the amount of ceramide in erythrocyte membranes. Both spectrin proteolysis and ceramide formation may cause the exposure of phosphatidylserine on the membrane so as to trigger a macrophage engulfment of the erythrocyte. By this means eryptosis may be an advantageous mechanism for removing defective erythrocytes before hemolysis. [ABSTRACT FROM AUTHOR]

Rodentibacter spp. are opportunistic pathogens that are often isolated from the upper respiratory tracts of laboratory rodents. In particular, R. pneumotropicus and R. heylii require considerable caution in rodent colonies, as they cause lethal pneumonia in rodents. A new species, R. haemolyticus , has recently been classified in the genus, and a very closely related strain, Rodentibacter sp. strain JRC, has been isolated in Japan. This study focused on strain JRC by performing genomic and pathogenic analyses. Draft genome sequencing of strain JRC identified several genes coding for putative virulent proteins, including hemolysin and adhesin. Furthermore, we found a new RTX (repeats-in-structural toxin) toxin gene in the genome, which was predicted to produce a critical virulence factor (RTXIA) similar to Enterobacteriaceae. The concentrated culture supernatant containing RTX toxin (RTXIA) showed cytotoxicity toward RAW264.7 cells. Pre-incubation with anti-CD11a attenuated the cytolysis, suggesting that the concentrated culture supernatant containing RTXIA is cell surface LFA-1 mediated cytolysin. Experimental infection of strain JRC intranasally with 5 female BALB/c- Rag2 −/− mice showed 60% lethality and was not significantly different from those of R. pneumotropicus ATCC 35149T using the log-rank test. Combined with our finding that RTXIA has an almost identical amino acid sequence (98% identity) to that of R. haemolyticus 1625/19T, these results strongly suggest that RTXIA-producing strain JRC (and related R. haemolyticus) is pathogenic to immunodeficient rodents, and both agents should be excluded in laboratory rodent colonies. • Rodentibacter sp. strain JRC and R. haemolyticus RTX toxin, which was similar to that produced by Enterobacteriaceae , was predicted an important virulence factor. • Concentrated culture supernatant containing RTX toxin cytolysis was attenuated by pre-incubation with anti-CD11a neutralizing antibody, suggesting that Rodentibacter sp. RTX toxin induces cell surface LFA-1 mediated cytolysins. • Experimental infection of Rodentibacter sp. intranasally with BALB/C- Rag2 −/− mice showed 60% lethality and was not significantly different from that of R. pneumotropicus type strain ATCC 35149T using the log-rank test. • RTX toxin producing Rodentibacter sp. strain JRC is one of agents that requires infection prevention in laboratory rodent colonies as well as R. haemolyticus. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic Escherichia coli (EHEC) cause diarrhea, bloody diarrhea and hemolytic-uremic syndrome (HUS), a thrombotic microangiopathy affecting the renal glomeruli, the intestine, and the brain. The pathogenesis of EHEC-mediated diseases is incompletely understood. In addition to Shiga toxins, the major virulence factors of EHEC, the contribution of EHEC hemolysin (EHEC-Hly), also designated EHEC toxin (Ehx), which is a member of the RTX (repeats-in-toxin) family, is increasingly recognized. The toxin and its activation and secretion machinery are encoded by the EHEC-hlyCABD operon, in which EHEC-hlyA is the structural gene for EHEC-Hly and the EHEC-hlyC product mediates post-translational activation of EHEC-Hly; the EHEC-hlyB- and EHEC-hlyD-encoded proteins form, together with genetically unlinked TolC, the type I secretion system that transports EHEC-Hly out of the bacterial cell. EHEC-Hly exists in two biologically active forms: as a free EHEC-Hly, and an EHEC-Hly associated with outer membrane vesicles (OMVs) that are released by EHEC during growth. The OMV-associated form results from a rapid binding of free EHEC-Hly to OMVs upon its extracellular secretion. The OMV association stabilizes EHEC-Hly and thus substantially prolongs its hemolytic activity compared to the free toxin. The two EHEC-Hly forms differ by their mechanism of toxicity toward human intestinal epithelial and microvascular endothelial cells, which are the major targets during EHEC infection. The free EHEC-Hly lyses human microvascular endothelial cells, presumably by pore formation in the cell membrane. In contrast, the OMV-associated EHEC-Hly does not lyse any of these cell types, but after its cellular internalization via OMVs it targets mitochondria and triggers caspase-9-mediated apoptosis. The proinflammatory potential of EHEC-Hly, in particular its ability to elicit secretion of interleukin-1β from human monocytes/macrophages, might be an additional mechanism of its putative contribution to the pathogenesis of EHEC-mediated diseases. Increasing understanding of molecular mechanisms underlying interaction of EHEC-Hly with target cells as well as the host cell responses to the toxin supports the involvement of EHEC-Hly in the pathogenesis of EHEC-mediated diseases and forms a basis for prevention of the EHEC-Hly-mediated injury during human infection. [ABSTRACT FROM AUTHOR]

Abstract: Avibacterium paragallinarum, the etiological agent of infectious coryza in chicken, was found to encode a bivalent serine-protease – RTX-porin toxin named AvxA. This toxin is encoded on a classical RTX operon structure with the activator gene avxC, the structural serin-protease-RTX toxin gene avxA, and the genes for a proper type I secretion system avxBD. AvxA is activated by the product of the avxC gene, secreted by the avxBD specified type I secretion system and proteolytically processed leaving a 95kDa RTX moiety that is found in culture supernatants of A. paragallinarum serovars A, B and C. The RTX moiety of AvxA (AvxA-RTX) is cytotoxic against the avian macrophage like cell line HD11 but not against bovine macrophage cell line BoMac. Purified IgG from hyper-immune rabbit anti-AvxA-RTX serum made by immunization with recombinant AvxA-RTX from a serotype A strain fully neutralizes the cytotoxic activity of recombinant active AvxA-RTX and of A. paragallinarum serotypes A, B and C. This indicates that AvxA is a common major virulence attribute of all A. paragallinarum serotypes. [Copyright &y& Elsevier]

Vibrio vulnificus, a halophilic estuarine bacterium causing fatal septicemia and necrotic wound infection, is highly cytotoxic to eukaryotic cells. We have reported that RtxA1 toxin kills host cells only after they come into contact with bacteria and plays an essential role in the pathogenesis of V. vulnificus. This study was performed to elucidate the mechanism by which the RtxA1 toxin mediates the death of HeLa cells. By using confocal microscopy and immunoblot analysis, we show that the 501-kDa RtxA1 toxin is processed into 2 fragments after its secretion into host cells. The largerN-terminal fragment (RtxA1-N; approximately 370 kDa) remained at the host cell membrane, whereas the smaller C-terminal fragment (RtxA1-C; approximately 130 kDa) was internalized into the host cell cytoplasm. RtxA1-N is believed to polymerize and form pores at the host cell membrane and to induce an increase in necrotic volume related to calcium. The RtxA1 toxin caused an increase in the intracellular Ca2+ concentration and the subsequent activation of JNK. The cell death mechanism occurred via calcium-dependent mitochondrial pathways, which caused calcium sequestration in the mitochondria, accompanied by irreversible mitochondrial membrane dysfunction and adenosine triphosphate depletion, and was later accompanied by the disruption of the integrity of the plasma membrane. [ABSTRACT FROM AUTHOR]

α-Hemolysin (HLY) is an important virulence factor for uropathogenic Escherichia coli. HLY is a member of the RTX family of exotoxins secreted by a number of Gram-negative bacteria. Recently, it was reported that a related RTX toxin, the Mannheimia haemolytica leukotoxin, exhibits increased cytotoxicity following brief heat treatment. In this article, we show that brief heat treatment (1 min at 100°C) increases cytotoxicity of HLY for human bladder cells, kidney epithelial cells (A498) and neutrophils. Heat treatment also increased hemolysis of human red blood cells (RBCs). Furthermore, heat treatment of previously inactived HLY restored its cytotoxicity. Heat-activated and native HLY both required glycophorin A to lyse RBCs. Native and heat-activated HLY appeared to bind equally well to the surface of A498 cells; although, Western blot analyses demonstrated binding to different proteins on the surface. Confocal microscopy revealed that heat-activated HLY bound more extensively to internal structures of permeabilized A498 cells than did native HLY. Several lines of spectroscopic evidence demonstrate irreversible changes in the structure of heat activated compared to native HLY. We show changes in secondary structure, increased exposure of tryptophan residues to the aqueous environment, an increase in molecular dimension and an increase in hydrophobic surface area. These properties are among the most common characteristics described for the molten globule state, first identified as an intermediate in protein folding. We hypothesize that brief heat treatment of HLY causes a conformational change leading to significant differences in protein-protein interactions that result in increased cytotoxicity for target cells. [ABSTRACT FROM AUTHOR]

The article reports on two cases of necrotizing mesenteric lymphangitis and lymphadenitis in foals with congenital hypothyroidism-dysmaturity syndrome (CHDS) and Actinobacillus suis-like organism (ASLO) sepsis. It outlines the pathologic and microbiologic findings in the two cases. It compares the results from the microbiologic, biochemical, protein and molecular biological analyses of the ASLOs isolated from the two foals with those of Actinobacillus isolates from foals and adult horses.