The Hunger Games: Aggregatibacter actinomycetemcomitans Exploits Human Neutrophils As an Epinephrine Source for Survival.

Aggregatibacter actinomycetemcomitans is a gram-negative facultative anaerobe and an opportunistic oral pathogen, strongly associated with periodontitis and other inflammatory diseases. Periodontitis is a chronic inflammation of the periodontium resulting from the inflammatory response of the host towards the dysbiotic microbial community present at the gingival crevice. Previously, our group identified catecholamines and iron as the signals that activate the QseBC two-component system in A. actinomycetemcomitans, necessary for the organism to acquire iron as a nutrient to survive in the anaerobic environment. However, the source of catecholamines has not been identified. It has been reported that mouse neutrophils can release catecholamines. In periodontitis, large infiltration of neutrophils is found at the subgingival pocket; hence, we wanted to test the hypothesis that A. actinomycetemcomitans exploits human neutrophils as a source for catecholamines. In the present study, we showed that human neutrophils synthesize, store, and release epinephrine, one of the three main types of catecholamines. Human neutrophil challenge with A. actinomycetemcomitans induced exocytosis of neutrophil granule subtypes: secretory vesicles, specific granules, gelatinase granules, and azurophilic granules. In addition, by selectively inhibiting granule exocytosis, we present the first evidence that epinephrine is stored in azurophilic granules. Using QseC mutants, we showed that the periplasmic domain of the QseC sensor kinase is required for the interaction between A. actinomycetemcomitans and epinephrine. Finally, epinephrine-containing supernatants collected from human neutrophils promoted A. actinomycetemcomitans growth and induced the expression of the qseBC operon under anaerobic conditions. Based on our findings, we propose that A. actinomycetemcomitans promotes azurophilic granule exocytosis by neutrophils as an epinephrine source to promote bacterial survival.

Transferrin Binding Protein B and Transferrin Binding Protein A2 Expand the Transferrin Recognition Range of Histophilus somni.

The bacterial bipartite transferrin receptor is an iron acquisition system that several important human and animal pathogens require for survival. It consists of the TonB-dependent transporter transferrin binding protein A (TbpA) and the surface lipoprotein transferrin binding protein B (TbpB). Curiously, the Tbps are only found in host-specific pathogens and are themselves host specific, meaning that they will bind to the transferrin of their host species but not to the transferrins of other animal species. While this phenomenon has long been established, neither the steps in the evolutionary process that led to this exquisite adaptation for the host nor the steps that could alter it are known. We sought to gain insight into these processes by studying Tbp specificity in Histophilus somni, an economically important pathogen of cattle. A past study showed that whole cells of H. somni specifically bind bovine transferrin but not transferrin from sheep and goats, two bovids whose transferrins share 93% amino acid sequence identity with bovine transferrin. To our surprise, we found that H. somni can use sheep and goat transferrins as iron sources for growth and that HsTbpB, but not HsTbpA, has detectable affinity for sheep and goat transferrins. Furthermore, a third transferrin binding protein found in H. somni, HsTbpA2, also showed affinity for sheep and goat transferrins. Our results suggest that H. somni TbpB and TbpA2 may contribute to broadening the host transferrin recognition range of H. somniIMPORTANCE Host-restricted pathogens infect a single host species or a narrow range of host species. Histophilus somni, a pathogen that incurs severe economic losses for the cattle industry, infects cattle, sheep, and goats but not other mammals. The transferrin binding proteins, TbpA and TbpB, are thought to be a key iron acquisition system in H. somni; however, despite their importance, H. somni TbpA and TbpB were previously shown to be cattle transferrin specific. In our study, we find that H. somni TbpB and another little-studied Tbp, TbpA2, bind sheep and goat transferrins, as well as bovine transferrin. Our results suggest that TbpB and TbpA2 may allow for host range expansion and provide a mechanism for how host specificity in Tbp-encoding pathogens can be altered.

Investigation of DNA Double-Strand Breaks Induced in Host Cells Following Infection with Genotoxic Bacteria.

Carcinogenesis is caused by genome instability, one of the major causes of which is double-strand DNA breaks (DSBs). Interestingly, infection by particular species of bacteria can induce DSBs in host cells. For example, several reports suggest an association between periodontal disease and oral cancer. Aggregatibacter actinomycetemcomitans, a common periodontal pathogen, causes DSBs in the host cell. Pulsed-field gel electrophoresis (PFGE) is often used to identify DSBs in host cells. However, as established during investigation of A. actinomycetemcomitans infection, it is often difficult to determine whether broken DNA fragments are indeed from human chromosomes or whether they are bacterial in origin using PFGE-based methods. Because the method involves the coculture of human cells with bacteria, both bacterial and human DNA fragments may be present in the broken DNA fraction. To address this problem, we have developed a method to detect only human chromosomal DNA upon PFGE analysis. Human chromosomes were prelabeled with halogenated deoxyuridine (e.g., BrdU and IdU) before being fractionated by PFGE and visualized by immunoblotting. As proof of concept, we successfully used this method to investigate the mechanism of DSB formation in host chromosomes following infection with genotoxic bacterial species.

Interactions between the Trimeric Autotransporter Adhesin EmaA and Collagen Revealed by Three-Dimensional Electron Tomography.

Bacterial adhesion to host tissues is considered the first and critical step of microbial infection. The extracellular matrix protein adhesin A (EmaA) is a collagen-binding adhesin of the periodontal pathogen Aggregatibacter actinomycetemcomitans Three 202-kDa EmaA monomers form antenna-like structures on the bacterial surface with the functional domain located at the apical end. The structure of the 30-nm functional domain has been determined by three-dimensional (3D) electron tomography and subvolume averaging. The region exhibits a complex architecture composed of three subdomains (SI to SIII) and a linker between subdomains SII and SIII. However, the molecular interaction between the adhesin receptor complexes has yet to be revealed. This study provides the first detailed 3D structure of reconstituted EmaA/collagen complexes obtained using 3D electron tomography and image processing techniques. The observed interactions of EmaA with collagen were not to whole, intact fibrils, but rather to individual collagen triple helices dissociated from the fibrils. The majority of the contacts with the EmaA functional domain encompassed subdomains SII and SIII and in some cases the tip of the apical domain, involving SI. These data suggest a multipronged mechanism for the interaction of Gram-negative bacteria with collagen.IMPORTANCE Bacterial adhesion is a crucial step for bacterial colonization and infection. In recent years, the number of antibiotic-resistant strains has dramatically increased; therefore, there is a need to search for novel antimicrobial agents. Thus, great efforts are being devoted to develop a clear understanding of the bacterial adhesion mechanism for preventing infections. In host/pathogen interactions, once repulsive forces are overcome, adhesins recognize and tightly bind to specific receptors on the host cell or tissue components. Here, we present the first 3D structure of the interaction between the collagen-binding adhesin EmaA and collagen, which is critical for the development of endocarditis in humans.

Differential quantitative proteomics study of experimental Mannheimia haemolytica mastitis in sheep.

Objective was the differential quantitative proteomics study of ovine mastitis induced by Mannheimia haemolytica; clinical, microbiological, cytological and histopathological methods were employed for confirmation and monitoring. Proteins were separated by two-dimensional gel electrophoresis (2-DE) for all samples and differentially abundant proteins were identified by mass spectrometry; comparisons were performed with pre- (blood, milk) and post- (milk of contralateral gland) inoculation findings. Animals developed mastitis, confirmed by isolation of challenge strain and increase of neutrophils in milk and by histopathological evidence. In blood plasma, 33 differentially abundant proteins (compared to findings before challenge) were identified: 6 with decrease, 13 with new appearance and 14 with varying abundance. In a post-challenge milk whey protein reference map, 65 proteins were identified; actin cytoplasmic-1, beta-lactoglobulin-1/B, cathelicidin-1 predominated. Further, 89 differentially abundant proteins (compared to findings before challenge) were identified: 18 with decrease, 53 with new appearance, 3 with increase and 15 with varying abundance; 15 proteins showed status changes in blood plasma and milk whey. Differential abundance from inoculated and contralateral glands revealed 74 proteins only from the inoculated gland. Most differentially abundant proteins in milk whey were involved in cell organisation and biogenesis (n = 17) or in inflammatory and defence response (n = 13). SIGNIFICANCE: The proteomes of blood and milk from ewes with experimental mastitis caused by Mannheimia haemolytica and the differential proteomics in sequential samples after challenge are presented for the first time. This is the first detailed proteomics study in M. haemolytica-associated mastitis in ewes. An experimental model fully simulating natural mastitis has been used. Use of experimentally induced mastitis minimised potential variations and allowed consistency of results. The study included evaluation of changes in blood plasma and milk whey. Protein patterns have been studied, indicating with great accuracy changes that had occurred as part of the disease process and development, during the acute phase of infection. Relevant protein-protein interactions were studied. The entirety of proteomics findings has suggested that affected ewes had mounted a defence response that had been regulated by many proteins (e.g., cathelicidins, haptoglobin, serum amyloid A) and through various pathways (e.g., acute phase response, binding and transporting significant ions and molecules); these were interdependent at various points. Potential biomarkers have been indicated for use in diagnostic assays of mastitis.

Novel Assay To Characterize Neutrophil Responses to Oral Biofilms.

Neutrophils, the most numerous leukocytes, play an important role in maintaining oral health through interactions with oral microbial biofilms. Both neutrophil hyperactivity and the bacterial subversion of neutrophil responses can cause inflammation-mediated tissue damage like that seen in periodontal disease. We describe here an assay that assesses neutrophil activation responses to monospecies biofilm bacteria in vitro based on the surface expression of cluster of differentiation (CD) markers associated with various neutrophil functions. Most of what we know about neutrophil responses to bacteria is based on in vitro assays that use planktonic bacteria and isolated/preactivated neutrophils, which makes interpretation of the neutrophil responses to bacteria a challenge. An understanding of how neutrophils differentially interact with and respond to commensal and pathogenic oral bacteria is necessary in order to further understand the neutrophil's role in maintaining oral health and the pathogenesis of periodontal disease. In this study, a flow cytometry-based in vitro assay was developed to characterize neutrophil activation states based on CD marker expressions in response to oral monospecies bacterial biofilms. Using this approach, changes in CD marker expressions in response to specific prominent oral commensal and pathogenic bacteria were assayed. Several functional assays, including assays for phagocytosis, production of reactive oxygen species, activation of the transcription factor Nrf2, neutrophil extracellular trap formation, and myeloperoxidase release, were also performed to correlate neutrophil function with CD marker expression. Our results demonstrate that neutrophils display bacterial species-specific responses. This assay can be used to characterize how specific biofilms alter specific neutrophil pathways associated with their activation.

Nephritis-associated plasmin receptor (NAPlr) positive glomerulonephritis caused by Aggregatibacter actinomycetemcomitans bacteremia: A case report.

Infection-related glomerulonephritis (IRGN) develops after various infections. It was previously thought to be caused by Streptococcus species alone but can also be caused by other pathogens. Nephritis-associated plasmin receptor (NAPlr) was discovered as a candidate nephritis-inducing factor in acute post-streptococcal glomerulonephritis. More recently, renal lesions caused by other pathogens were found to be positive for the same molecular marker. We report the case of a 64-year-old man who experienced repeated fever for several months and presented with progressively-deteriorating renal function. He had previously undergone aortic valve replacement. Aggregatibacter actinomycetemcomitans, a component of the oral flora, was detected in a blood culture. Renal biopsy showed diffuse proliferative glomerulonephritis. Immunofluorescence staining of the kidney specimen was positive for immunoglobulins, complements, and NAPlr. The patient was diagnosed with infectious endocarditis and IRGN. Six weeks of intravenous antibiotic therapy improved the patient's clinical condition and kidney function. In this case, IRGN was caused by a rare pathogen. This is the first published case to show NAPlr positivity in the glomeruli after systemic infection with the periodontal bacteria, Aggregatibacter actinomycetemcomitans. This case and subsequent research might expand the concept of IRGN, anchored by NAPlr as a key diagnostic biomarker.
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Aggregatibacter actinomycetemcomitans regulates the expression of integrins and reduces cell adhesion via integrin α5 in human gingival epithelial cells.

Gingival epithelial cells form a physiological barrier against bacterial invasion. Excessive bacterial invasion destroys the attachment between the tooth surface and the epithelium, resulting in periodontitis. Integrins play a significant role in cell attachment; therefore, we hypothesized that bacterial infection might decrease the expressions of these integrins in gingival epithelial cells, resulting in reduced cell adhesion. Immortalized human gingival epithelial cells were co-cultured with Aggregatibacter actinomycetemcomitans Y4 (Aa Y4), and the gene expression levels of IL-8, proliferating cell nuclear antigen (PCNA), and integrins (α2, α3, α5, ß4, and ß6) were measured using quantitative reverse transcription polymerase chain reaction. Expression of PCNA and integrins, except integrin α5, was significantly downregulated, while expression of IL-8 and integrin α5 was significantly upregulated in the cells co-cultured with Aa Y4. The number of adherent cells significantly decreased when co-cultured with Aa Y4, as determined using cell adhesion assays. In the cells co-cultured with Aa Y4 and an integrin α5 neutralizing antibody, there was no effect on the expression of IL-8 and PCNA, while the expressions of integrins α2, α3, ß4, and ß6, and the number of adherent cells did not decrease. The number of invading bacteria in the cells was reduced in the presence of the antibody and increased in the presence of TLR2/4 inhibitor. Therefore, integrin α5 might be involved in Aa Y4 invasion into gingival epithelial cells, and the resulting signal transduction cascade reduces cell adhesion by decreasing the expression of integrins, while the TLR2/4 signaling cascade regulates IL-8 expression.

Localization and expression pattern of amelotin, odontogenic ameloblast-associated protein and follicular dendritic cell-secreted protein in the junctional epithelium of inflamed gingiva.

The purpose of this study is to elucidate the localization of amelotin (AMTN), odontogenic ameloblast-associated protein (ODAM) and follicular dendritic cell-secreted protein (FDC-SP) at the junctional epithelium (JE) in Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans infected mice and inflamed and non-inflamed human gingiva. We performed immunostaining to determine the localization and expression pattern of AMTN, ODAM and FDC-SP. AMTN, ODAM and FDC-SP in A. actinomycetemcomitans infected mice did not change dramatically compared with non-infected mice. AMTN and FDC-SP expressions were observed stronger in P. gingivalis infected mice at early stage. However, at the following stage, the coronal part of the AMTN expression disappeared from the JE, and FDC-SP expression decreased due to severe inflammation by P. gingivalis. ODAM expressed internal and external basal lamina, and the expression increased not only at early stage but also at the following stage in the inflammatory JE induced by P. gingivalis. In the human gingival tissues, AMTN was detected at the surface of the sulcular epithelium and JE in the non-inflamed and inflamed gingiva, and the localization did not change the process of inflammation. ODAM and FDC-SP were more widely detected at the sulcular epithelium and JE in the non-inflamed gingiva. In the inflamed gingiva, localization of ODAM and FDC-SP was spread into the gingival epithelium, compared to AMTN. These studies demonstrated that the expression pattern of AMTN, ODAM and FDC-SP at the JE were changed during inflammation process and these three proteins might play an important role in the resistance to inflammation.

Mitogen-Activated Protein Kinase 2 Signaling Shapes Macrophage Plasticity in Aggregatibacter actinomycetemcomitans-Induced Bone Loss.

Aggregatibacter actinomycetemcomitans is associated with aggressive periodontal disease, which is characterized by inflammation-driven alveolar bone loss. A. actinomycetemcomitans activates the p38 mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase 2 (MK2) stress pathways in macrophages that are involved in host responses. During the inflammatory process in periodontal disease, chemokines are upregulated to promote recruitment of inflammatory cells. The objective of this study was to determine the role of MK2 signaling in chemokine regulation during A. actinomycetemcomitans pathogenesis. Utilizing a murine calvarial model, Mk2+/+ and Mk2-/- mice were treated with live A. actinomycetemcomitans bacteria at the midsagittal suture. MK2 positively regulated the following macrophage RNA: Emr1 (F4/80), Itgam (CD11b), Csf1r (M-CSF Receptor), Itgal (CD11a), Tnf, and Nos2 Additionally, RNA analysis revealed that MK2 signaling regulated chemokines CCL3 and CCL4 in murine calvarial tissue. Utilizing the chimeric murine air pouch model, MK2 signaling differentially regulated CCL3 and CCL4 in the hematopoietic and nonhematopoietic compartments. Bone resorption pits in calvaria, observed by micro-computed tomography, and osteoclast formation were decreased in Mk2-/- mice compared to Mk2+/+ mice after A. actinomycetemcomitans treatment. In conclusion, these data suggest that MK2 in macrophages contributes to regulation of chemokine signaling during A. actinomycetemcomitans-induced inflammation and bone loss.

Microbial Community Composition Impacts Pathogen Iron Availability during Polymicrobial Infection.

Iron is an essential nutrient for bacterial pathogenesis, but in the host, iron is tightly sequestered, limiting its availability for bacterial growth. Although this is an important arm of host immunity, most studies examine how bacteria respond to iron restriction in laboratory rather than host settings, where the microbiome can potentially alter pathogen strategies for acquiring iron. One of the most important transcriptional regulators controlling bacterial iron homeostasis is Fur. Here we used a combination of RNA-seq and chromatin immunoprecipitation (ChIP)-seq to characterize the iron-restricted and Fur regulons of the biofilm-forming opportunistic pathogen Aggregatibacter actinomycetemcomitans. We discovered that iron restriction and Fur regulate 4% and 3.5% of the genome, respectively. While most genes in these regulons were related to iron uptake and metabolism, we found that Fur also directly regulates the biofilm-dispersing enzyme Dispersin B, allowing A. actinomycetemcomitans to escape from iron-scarce environments. We then leveraged these datasets to assess the availability of iron to A. actinomycetemcomitans in its primary infection sites, abscesses and the oral cavity. We found that A. actinomycetemcomitans is not restricted for iron in a murine abscess mono-infection, but becomes restricted for iron upon co-infection with the oral commensal Streptococcus gordonii. Furthermore, in the transition from health to disease in human gum infection, A. actinomycetemcomitans also becomes restricted for iron. These results suggest that host iron availability is heterogeneous and dependent on the infecting bacterial community.

Withaferin A inhibits inflammatory responses induced by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in macrophages.

Periodontitis is a progressive chronic inflammatory disease and a major cause of tooth loss in humans. As a withanolides, withaferin A (WA) is known to exhibit strong anti­inflammatory activity. The present study examined whether WA inhibited inflammatory responses in macrophages in response to two representative periodontal pathogens, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. Murine bone marrow­derived macrophages (BMDMs) were used in this study and cytokine production in culture supernatants was measured by enzyme­linked immunosorbent assays. Western blot analysis was performed to determine the activation of nuclear factor­κB and mitogen­activated protein kinases (MAPKs) and the expression of inducible nitric oxide synthase (iNOS), toll­like receptor (TLR) 2 and TLR4. The production of nitric oxide (NO) was determined by the Griess reaction. WA treatment was shown to decrease interleukin (IL)­6 and tumor necrosis factor (TNF)­α production in BMDMs in response to F. nucleatum and A. actinomycetemcomitans in a dose­dependent manner. The phosphorylation of IκB­α and MAPKs (p38, extracellular signal­regulated kinases and c­Jun N­terminal kinases) induced by F. nucleatum and A. actinomycetemcomitans was also inhibited by WA. F. nucleatum and A. actinomycetemcomitans induced iNOS expression and NO production in BMDMs, which was inhibited by WA in a dose­dependent manner. WA also reduced endogenous and induced expression of TLR2 and TLR4 in these cells. These results suggest that WA may be a potential therapeutic agent or preventive additive for periodontitis control.

Interleukin-1 Receptor Antagonist Has a Novel Function in the Regulation of Matrix Metalloproteinase-13 Expression.

Interleukin-1 receptor antagonist (IL-1Ra) is an IL-1 family member, which binds to IL-1 receptors but does not induce any intracellular signaling. We addressed whether IL-1Ra has a novel function in regulation of the extracellular matrix or adhesion molecules. Polymerase chain reaction array analysis demonstrated a ~5-fold increase in matrix metalloproteinase 13 (MMP-13) mRNA expression of IL-1Ra siRNA-transfected Ca9-22 human oral squamous epithelial carcinoma cells compared with the control. In fact, MMP-13 mRNA and protein expression as well as its activity in IL-1Ra siRNA-transfected Ca9-22 cell lines were significantly higher than those in the control. IL-1Ra siRNA treatment resulted in strong elevation of MMP-13 expression, whereas addition of rhIL-1Ra (40 ng/ml) suppressed MMP-13 expression, suggesting that IL-1Ra had a specific effect on MMP-13 induction. IL-1Ra siRNA could potently suppress IL-1α. No significant difference was found between the MMP-13 mRNA expression of IL-1Ra siRNA-transfected cells and those treated with anti-IL-1α or anti-IL-1ß antibodies. These results suggested that continuous supply of IL-1 had no effect on the induction of MMP-13 by IL-1Ra siRNA. Histopathological investigation of MMP-13 in periodontal tissue showed specific localization in the junctional epithelial cells of IL-1Ra knockout (KO) mice. Furthermore, infection with Aggregatibacter actinomycetemcomitans to establish an experimental periodontitis model resulted in predominant localization of MMP-13 along apical junctional epithelial cells. Laminin-5, which is degraded by MMP-13, was found in the internal basal lamina of wild-type mice, whereas the internal basal lamina of IL-1Ra KO mice did not show obvious laminin-5 localization. In particular, laminin-5 localization almost disappeared in the internal basal lamina of IL-1Ra KO mice infected with A. actinomycetemcomitans, suggesting that the suppression of IL-1Ra resulted in strong induction of MMP-13 that degraded laminin-5. In conclusion, IL-1Ra is associated with MMP-13 expression and has a novel function in such regulation without interference of the IL-1 signaling cascade.

The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Active Subunit CdtB Contains a Cholesterol Recognition Sequence Required for Toxin Binding and Subunit Internalization.

Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1ß [IL-1ß] and tumor necrosis factor alpha [TNF-α] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.

Aggregatibacter actinomycetemcomitans leukotoxin (LtxA; Leukothera) induces cofilin dephosphorylation and actin depolymerization during killing of malignant monocytes.

Leukotoxin (LtxA; Leukothera), a protein toxin secreted by the oral bacterium Aggregatibacter actinomycetemcomitans, specifically kills white blood cells (WBCs). LtxA binds to the receptor known as lymphocyte function associated antigen-1 (LFA-1), a ß2 integrin expressed only on the surface of WBCs. LtxA is being studied as a virulence factor that helps A. actinomycetemcomitans evade host defences and as a potential therapeutic agent for the treatment of WBC diseases. LtxA-mediated cell death in monocytes involves both caspases and lysosomes; however, the signalling proteins that regulate and mediate cell death remain largely unknown. We used a 2D-gel proteomics approach to analyse the global protein expression changes that occur in response to LtxA. This approach identified the protein cofilin, which underwent dephosphorylation upon LtxA treatment. Cofilin is a ubiquitous actin-binding protein known to regulate actin dynamics and is regulated by LIM kinase (LIMK)-mediated phosphorylation. LtxA-mediated cofilin dephosphorylation was dependent on LFA-1 and cofilin dephosphorylation did not occur when LFA-1 bound to its natural ligand, ICAM-1. Treatment of cells with an inhibitor of LIMK (LIMKi) also led to cofilin dephosphorylation and enhanced killing by LtxA. This enhanced sensitivity to LtxA coincided with an increase in lysosomal disruption, and an increase in LFA-1 surface expression and clustering. Both LIMKi and LtxA treatment also induced actin depolymerization, which could play a role in trafficking and surface distribution of LFA-1. We propose a model in which LtxA-mediated cofilin dephosphorylation leads to actin depolymerization, LFA-1 overexpression/clustering, and enhanced lysosomal-mediated cell death.

Diverse Toll-like receptors mediate cytokine production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in macrophages.

Toll-like receptors (TLRs) orchestrate a repertoire of immune responses in macrophages against various pathogens. Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans are two important periodontal pathogens. In the present study, we investigated TLR signaling regulating cytokine production of macrophages in response to F. nucleatum and A. actinomycetemcomitans. TLR2 and TLR4 are redundant in the production of cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) in F. nucleatum- and A. actinomycetemcomitans-infected macrophages. The production of cytokines by macrophages in response to F. nucleatum and A. actinomycetemcomitans infection was impaired in MyD88-deficient macrophages. Moreover, cytokine concentrations were lower in MyD88-deficient macrophages than in TLR2/TLR4 (TLR2/4) double-deficient cells. An endosomal TLR inhibitor, chloroquine, reduced cytokine production in TLR2/4-deficient macrophages in response to F. nucleatum and A. actinomycetemcomitans, and DNA from F. nucleatum or A. actinomycetemcomitans induced IL-6 production in bone marrow-derived macrophages (BMDMs), which was abolished by chloroquine. Western blot analysis revealed that TLR2/4 and MyD88 were required for optimal activation of NF-κB and mitogen-activated protein kinases (MAPKs) in macrophages in response to F. nucleatum and A. actinomycetemcomitans, with different kinetics. An inhibitor assay showed that NF-κB and all MAPKs (p38, extracellular signal-regulated kinase [ERK], and Jun N-terminal protein kinase [JNK]) mediate F. nucleatum-induced production of cytokines in macrophages, whereas NF-κB and p38, but not ERK and JNK, are involved in A. actinomycetemcomitans-mediated cytokine production. These findings suggest that multiple TLRs may participate in the cytokine production of macrophages against periodontal bacteria.

Bacterial infection increases periodontal bone loss in diabetic rats through enhanced apoptosis.

Periodontal disease is the most common osteolytic disease in humans and is significantly increased by diabetes mellitus. We tested the hypothesis that bacterial infection induces bone loss in diabetic animals through a mechanism that involves enhanced apoptosis. Type II diabetic rats were inoculated with Aggregatibacter actinomycetemcomitans and treated with a caspase-3 inhibitor, ZDEVD-FMK, or vehicle alone. Apoptotic cells were measured with TUNEL; osteoblasts and bone area were measured in H&E sections. New bone formation was assessed by labeling with fluorescent dyes and by osteocalcin mRNA levels. Osteoclast number, eroded bone surface, and new bone formation were measured by tartrate-resistant acid phosphatase staining. Immunohistochemistry was performed with an antibody against tumor necrosis factor-α. Bacterial infection doubled the number of tumor necrosis factor-α-expressing cells and increased apoptotic cells adjacent to bone 10-fold (P < 0.05). Treatment with caspase inhibitor blocked apoptosis, increased the number of osteoclasts, and eroded bone surface (P < 0.05); yet, inhibition of apoptosis resulted in significantly greater net bone area because of an increase in new bone formation, osteoblast numbers, and an increase in bone coupling. Thus, bacterial infection in diabetic rats stimulates periodontitis, in part through enhanced apoptosis of osteoblastic cells that reduces osseous coupling through a caspase-3-dependent mechanism.

Bovine respiratory syncytial virus and Histophilus somni interaction at the alveolar barrier.

Our previous studies showed that Histophilus somni and bovine respiratory syncytial virus (BRSV) act synergistically in vivo to cause more severe bovine respiratory disease than either agent alone causes. Since H. somni surface and secreted immunoglobulin binding protein A (IbpA) causes retraction of bovine alveolar type 2 (BAT2) cells and invasion between BAT2 cells in vitro, we investigated mechanisms of BRSV-plus-H. somni infection at the alveolar barrier. BRSV treatment of BAT2 cells prior to treatment with IbpA-rich H. somni concentrated culture supernatant (CCS) resulted in increased BAT2 cell rounding and retraction compared to those with either treatment alone. This mimicked the increased alveolar cell thickening in calves experimentally infected with BRSV followed by H. somni compared to that in calves infected with BRSV or H. somni alone. BRSV-plus-H. somni CCS treatment of BAT2 cells also enhanced paracellular migration. The effect of matrix metalloproteinases (MMPs) was investigated as well because microarray analysis revealed that treatment with BRSV plus H. somni synergistically upregulated BAT2 cell expression of mmp1 and mmp3 compared to that in cells treated with either agent alone. Enzyme-linked immunosorbent assay (ELISA) confirmed that MMP1 and MMP3 protein levels were similarly upregulated. In collagen I and collagen IV (targets for MMP1 and MMP3, respectively) substrate zymography, digestion was increased with supernatants from dually treated BAT2 cells compared with those from singly treated cells. Enhanced breakdown of collagen IV in the basal lamina and of fibrillar collagen I in the adjacent interstitium in the dual infection may facilitate dissemination of H. somni infection.

Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: interactions with human ß2 integrins and erythrocytes.

Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a frequent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin (RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear phagocytes. The known receptor for LtxA on leukocytes is integrin α(L)ß(2) (LFA-1 or CD11a/CD18). However, the molecular mechanisms involved in LtxA-mediated cytotoxicity are poorly understood, partly because LtxA has proven difficult to prepare for experiments as free of contaminants and with its native structure. Here, we describe a protocol for the purification of LtxA from bacterial culture supernatant, which does not involve denaturing procedures. The purified LtxA was monodisperse, well folded as judged by the combined use of synchrotron radiation circular dichroism spectroscopy (SRCD) and in silico prediction of the secondary structure content, and free of bacterial lipopolysaccharide. The analysis by SRCD and similarity to a lipase from Pseudomonas with a known three dimensional structure supports the presence of a so-called beta-ladder domain in the C-terminal part of LtxA. LtxA rapidly killed K562 target cells transfected to express ß(2) integrin. Cells expressing α(M)ß(2) (CD11b/CD18) or α(X)ß(2) (CD11c/CD18) were killed as efficiently as cells expressing α(L)ß(2). Erythrocytes, which do not express ß(2) integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the ß(2) chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins α(M)ß(2) and α(X)ß(2) as novel receptors for LtxA.

Effects of exposure to calves persistently infected with bovine viral diarrhea virus type 1b and Mannheimia haemolytica challenge on animal performance, nitrogen balance, and visceral organ mass in beef steers.

Bovine viral diarrhea viruses (BVDV) have been isolated alone or in combination with other viral and bacterial pathogens in animals diagnosed with bovine respiratory disease (BRD), a disease causing major economic loss to the feedlot industry. The objective of this experiment was to determine the effects of Mannheimia haemolytica challenge after short-term exposure (72 h) to bovine viral diarrhea virus type 1b (BVDV1b) persistently infected (PI) calves on performance, N balance, and organ mass in finishing cattle. Treatments (6 steers/treatment; initial BW = 314 +/- 31 kg) were 1) steers not exposed to steers PI with BVDV nor challenged with M. haemolytica (control; CON); 2) steers exposed to 2 steers PI with BVDV1b (BVD) for 72 h; 3) steers intratracheally challenged with M. haemolytica (MH); or 4) steers exposed to 2 steers PI with BVDV1b for 72 h and challenged with M. haemolytica (BVD+MH). There were 12 h between exposure to PI steers and challenge with M. haemolytica. Steers were housed in metabolism stanchions during the first 5 d after the M. haemolytica challenge and on d 7 to 11, 28 to 32, and for 5 d before slaughter (average 119 d on feed) to determine N balance and were weighed every 28 d. At slaughter, carcass and organ mass data were collected. Data were analyzed as a randomized complete block design with a 2 x 2 factorial arrangement of treatments, and steer was used as the experimental unit. From d -3 (beginning of PI steer exposure) to 4, steers challenged with M. haemolytica had less (P = 0.04) ADG than steers not challenged with M. haemolytica. In addition, steers exposed to steers PI with BVDV tended (P = 0.09) to have less ADG and G:F across the entire finishing period than steers not exposed to BVDV. Before slaughter, retained N expressed as grams per day (P = 0.03) and as a percentage of N intake (P = 0.04) was less in BVD steers compared with steers not exposed to BVDV. There were no effects (P > 0.10) of BVDV exposure or M. haemolytica challenge on empty BW (EBW) or carcass characteristics. Expressed as a percentage of EBW, HCW was less (P = 0.02) and total offal weight was greater (P = 0.02) for steers challenged with M. haemolytica compared with steers not challenged. Results are in agreement with those reported in larger scale finishing studies and suggest that acute exposure to BRD-related pathogens can have long-term effects on animal performance.