Emergence of Erythromycin-Resistant Invasive Group A Streptococcus, West Virginia, USA, 2020-2021.

Clindamycin and ß-lactam antibiotics have been mainstays for treating invasive group A Streptococcus (iGAS) infection, yet such regimens might be limited for strains displaying MLSB phenotypes. We investigated 76 iGAS isolates from 66 patients in West Virginia, USA, during 2020-2021. We performed emm typing using Centers for Disease Control and Prevention guidelines and assessed resistance both genotypically and phenotypically. Median patient age was 42 (range 23-86) years. We found 76% of isolates were simultaneously resistant to erythromycin and clindamycin, including all emm92 and emm11 isolates. Macrolide resistance was conferred by the plasmid-borne ermT gene in all emm92 isolates and by chromosomally encoded ermA, ermB, and a single mefA in other emm types. Macrolide-resistant iGAS isolates were typically resistant to tetracycline and aminoglycosides. Vulnerability to infection was associated with socioeconomic status. Our results show a predominance of macrolide-resistant isolates and a shift in emm type distribution compared with historical reports.

Prevalence of erythromycin-resistant emm92-type invasive group A streptococcal infections among injection drug users in West Virginia, United States, 2021-23.

BACKGROUND: Increasing incidence of invasive group A Streptococcus (iGAS) disease has been reported in Europe and the USA over the past several years. Coupled with this are observations of higher rates of resistance to erythromycin and clindamycin. OBJECTIVES: To characterize iGAS and pharyngitis isolates from West Virginia (WV), a US state outside of the national Active Bacteria Core surveillance purview, where risk factors associated with iGAS infections are prevalent. METHODS: Seventy-seven invasive group A Streptococcus isolates were collected from 67 unique patients at the J.W. Ruby Memorial Hospital Clinical Microbiology Laboratory in WV from 2021 to 2023. Invasive isolates and 20 unique pharyngitis isolates were tested for clindamycin and erythromycin susceptibility in the clinical laboratory. Patient demographic and clinical information was retrieved from patient electronic health records. Isolates were further characterized based on emm subtype and detection of MLSB resistance determinants. RESULTS: Twenty-six (39%) isolates were of a single emm92 type. All emm92 isolates were uniformly erythromycin/clindamycin resistant with inducible or constitutive MLSB resistance imparted by the plasmid-borne erm(T) gene. The majority of emm92 infections were associated with adult patients who reported IV drug use, whereas no pharyngitis infections were caused by an emm92 strain. Overall, 51 (76%) of the 67 iGAS isolates were determined to carry MLSB resistance. CONCLUSIONS: Isolates of emm92 type (clonal subtype emm92.0) were associated with iGAS infections in adult IV drug users, but not with paediatric pharyngitis, and were uniformly resistant to erythromycin and clindamycin.

Adaptation of the group A Streptococcus adhesin Scl1 to bind fibronectin type III repeats within wound-associated extracellular matrix: implications for cancer therapy.

The human-adapted pathogen group A Streptococcus (GAS) utilizes wounds as portals of entry into host tissue, wherein surface adhesins interact with the extracellular matrix, enabling bacterial colonization. The streptococcal collagen-like protein 1 (Scl1) is a major adhesin of GAS that selectively binds to two fibronectin type III (FnIII) repeats within cellular fibronectin, specifically the alternatively spliced extra domains A and B, and the FnIII repeats within tenascin-C. Binding to FnIII repeats was mediated through conserved structural determinants present within the Scl1 globular domain and facilitated GAS adherence and biofilm formation. Isoforms of cellular fibronectin that contain extra domains A and B, as well as tenascin-C, are present for several days in the wound extracellular matrix. Scl1-FnIII binding is therefore an example of GAS adaptation to the host's wound environment. Similarly, cellular fibronectin isoforms and tenascin-C are present in the tumor microenvironment. Consistent with this, FnIII repeats mediate GAS attachment to and enhancement of biofilm formation on matrices deposited by cancer-associated fibroblasts and osteosarcoma cells. These data collectively support the premise for utilization of the Scl1-FnIII interaction as a novel method of anti-neoplastic targeting in the tumor microenvironment.

Surface-exposed loops and an acidic patch in the Scl1 protein of group A Streptococcus enable Scl1 binding to wound-associated fibronectin.

Keratinized epidermis constitutes a powerful barrier of the mucosa and skin, effectively preventing bacterial invasion, unless it is wounded and no longer protective. Wound healing involves deposition of distinct extracellular matrix (ECM) proteins enriched in cellular fibronectin (cFn) isoforms containing extra domain A (EDA). The streptococcal collagen-like protein 1 (Scl1) is a surface adhesin of group A Streptococcus (GAS), which contains an N-terminal variable (V) domain and a C-terminally located collagen-like domain. During wound infection, Scl1 selectively binds EDA/cFn isoforms and laminin, as well as low-density lipoprotein (LDL), through its V domain. The trimeric V domain has a six-helical bundle fold composed of three pairs of anti-parallel α-helices interconnected by hypervariable loops, but the roles of these structures in EDA/cFn binding are unclear. Here, using recombinant Scl (rScl) constructs to investigate structure-function determinants of the Scl1-EDA/cFn interaction, we found that full-length rScl1, containing both the globular V and the collagen domains, is necessary for EDA/cFn binding. We established that the surface-exposed loops, interconnecting conserved α-helices, guide recognition and binding of Scl1-V to EDA and binding to laminin and LDL. Moreover, electrostatic surface potential models of the Scl1-V domains pointed to a conserved, negatively charged pocket, surrounded by positively charged and neutral regions, as a determining factor for the binding. In light of these findings, we propose an updated model of EDA/cFn recognition by the Scl1 adhesin from GAS, representing a significant step in understanding the Scl1-ECM interactions within the wound microenvironment that underlie GAS pathogenesis.

Collagen-like proteins of pathogenic streptococci.

The collagen domain, which is defined by the presence of the Gly-X-Y triplet repeats, is amongst the most versatile and widespread known structures found in proteins from organisms representing all three domains of life. The streptococcal collagen-like (Scl) proteins are widely present in pathogenic streptococci, including Streptococcus pyogenes, S. agalactiae, S. pneumoniae, and S. equi. Experiments and bioinformatic analyses support the hypothesis that all Scl proteins are homotrimeric and cell wall-anchored. These proteins contain the rod-shaped collagenous domain proximal to cell surface, as well as a variety of outermost non-collagenous domains that generally lack predicted functions but can be grouped into one of six clusters based on sequence similarity. The well-characterized Scl1 proteins of S. pyogenes show a dichotomous switch in ligand binding between human tissue and blood environments. In tissue, Scl1 adhesin specifically recognizes the wound microenvironment, promotes adhesion and biofilm formation, decreases bacterial killing by neutrophil extracellular traps, and modulates S. pyogenes virulence. In blood, ligands include components of complement and coagulation-fibrinolytic systems, as well as plasma lipoproteins. In all, the Scl proteins signify a large family of structurally related surface proteins, which contribute to the ability of streptococci to colonize and cause diseases in humans and animals.

The crystal structure of the streptococcal collagen-like protein 2 globular domain from invasive M3-type group A Streptococcus shows significant similarity to immunomodulatory HIV protein gp41.

The arsenal of virulence factors deployed by streptococci includes streptococcal collagen-like (Scl) proteins. These proteins, which are characterized by a globular domain and a collagen-like domain, play key roles in host adhesion, host immune defense evasion, and biofilm formation. In this work, we demonstrate that the Scl2.3 protein is expressed on the surface of invasive M3-type strain MGAS315 of Streptococcus pyogenes. We report the crystal structure of Scl2.3 globular domain, the first of any Scl. This structure shows a novel fold among collagen trimerization domains of either bacterial or human origin. Despite there being low sequence identity, we observed that Scl2.3 globular domain structurally resembles the gp41 subunit of the envelope glycoprotein from human immunodeficiency virus type 1, an essential subunit for viral fusion to human T cells. We combined crystallographic data with modeling and molecular dynamics techniques to gather information on the entire lollipop-like Scl2.3 structure. Molecular dynamics data evidence a high flexibility of Scl2.3 with remarkable interdomain motions that are likely instrumental to the protein biological function in mediating adhesive or immune-modulatory functions in host-pathogen interactions. Altogether, our results provide molecular tools for the understanding of Scl-mediated streptococcal pathogenesis and important structural insights for the future design of small molecular inhibitors of streptococcal invasion.

Natural variant of collagen-like protein a in serotype M3 group a Streptococcus increases adherence and decreases invasive potential.

Group A Streptococcus (GAS) predominantly exists as a colonizer of the human oropharynx that occasionally breaches epithelial barriers to cause invasive diseases. Despite the frequency of GAS carriage, few investigations into the contributory molecular mechanisms exist. To this end, we identified a naturally occurring polymorphism in the gene encoding the streptococcal collagen-like protein A (SclA) in GAS carrier strains. All previously sequenced invasive serotype M3 GAS possess a premature stop codon in the sclA gene truncating the protein. The carrier polymorphism is predicted to restore SclA function and was infrequently identified by targeted DNA sequencing in invasive strains of the same serotype. We demonstrate that a strain with the carrier sclA allele expressed a full-length SclA protein, while the strain with the invasive sclA allele expressed a truncated variant. An isoallelic mutant invasive strain with the carrier sclA allele exhibited decreased virulence in a mouse model of invasive disease and decreased multiplication in human blood. Further, the isoallelic invasive strain with the carrier sclA allele persisted in the mouse nasopharynx and had increased adherence to cultured epithelial cells. Repair of the premature stop codon in the invasive sclA allele restored the ability to bind the extracellular matrix proteins laminin and cellular fibronectin. These data demonstrate that a mutation in GAS carrier strains increases adherence and decreases virulence and suggest selection against increased adherence in GAS invasive isolates.

The group A streptococcal collagen-like protein-1, Scl1, mediates biofilm formation by targeting the extra domain A-containing variant of cellular fibronectin expressed in wounded tissue.

Wounds are known to serve as portals of entry for group A Streptococcus (GAS). Subsequent tissue colonization is mediated by interactions between GAS surface proteins and host extracellular matrix components. We recently reported that the streptococcal collagen-like protein-1, Scl1, selectively binds the cellular form of fibronectin (cFn) and also contributes to GAS biofilm formation on abiotic surfaces. One structural feature of cFn, which is predominantly expressed in response to tissue injury, is the presence of a spliced variant containing extra domain A (EDA/EIIIA). We now report that GAS biofilm formation is mediated by the Scl1 interaction with EDA-containing cFn. Recombinant Scl1 proteins that bound cFn also bound recombinant EDA within the C-C' loop region recognized by the α(9)ß(1) integrin. The extracellular 2-D matrix derived from human dermal fibroblasts supports GAS adherence and biofilm formation. Altogether, this work identifies and characterizes a novel molecular mechanism by which GAS utilizes Scl1 to specifically target an extracellular matrix component that is predominantly expressed at the site of injury in order to secure host tissue colonization.

Group A Streptococcal Collagen-like Protein 1 Restricts Tumor Growth in Murine Pancreatic Adenocarcinoma and Inhibits Cancer-Promoting Neutrophil Extracellular Traps.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer associated with an immunosuppressive environment. Neutrophil extracellular traps (NETs) were initially described in the context of infection but have more recently been implicated in contributing to the tolerogenic immune response in PDAC. Thus, NETs are an attractive target for new therapeutic strategies. Group A Streptococcus (GAS) has developed defensive strategies to inhibit NETs. In the present work, we propose utilizing intra-tumoral GAS injection to stimulate anti-tumor activity by inhibiting cancer-promoting NETs. Injection of three different M-type GAS strains reduced subcutaneous pancreatic tumor volume compared to control in two different murine PDAC models. Limitation of tumor growth was dependent on streptococcal collagen-like protein 1 (Scl1), as isogenic mutant strain devoid of Scl1 did not reduce tumor size. We further show that Scl1 plays a role in localizing GAS to the tumor site, thereby limiting the systemic spread of bacteria and off-target effects. While mice did elicit a humoral immune response to GAS antigens, tested sera were negative toward Scl1 antigen following intra-tumoral treatment with Scl1-expressing GAS. M1 GAS inhibited NET formation when co-cultured with neutrophils while Scl1-devoid mutant strain did not. Recombinant Scl1 protein inhibited NETs ex vivo in a dose-dependent manner by suppressing myeloperoxidase activity. Altogether, we demonstrate that intra-tumoral GAS injections reduce PDAC growth, which is facilitated by Scl1, in part through inhibition of cancer promoting NETs. This work offers a novel strategy by which NETs can be targeted through Scl1 protein and potentiates its use as a cancer therapeutic.

Group A streptococcal collagen-like protein 1 restricts tumor growth in murine pancreatic adenocarcinoma and inhibits cancer-promoting neutrophil extracellular traps.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer associated with an immunosuppressive environment. Neutrophil extracellular traps (NETs) were initially described in the context of infection but have more recently been implicated in contributing to the tolerogenic immune response in PDAC. Thus, NETs are an attractive target for new therapeutic strategies. Group A Streptococcus (GAS) has developed defensive strategies to inhibit NETs. Methods: In the present work, we propose utilizing intra-tumoral GAS injection to stimulate anti-tumor activity by inhibiting cancer-promoting NETs. Mice harboring Panc02 or KPC subcutaneous tumors injected with three different M-type GAS strains. Tumors and spleens were harvested at the endpoint of the experiments to assess bacterial colonization and systemic spread, while sera were analyzed for humoral responses toward the streptococcal antigens, especially the M1 and Scl1 proteins. Role of the streptococcal collagen-like protein 1 (Scl1) in anti-PDAC activity was assessed in vivo after intratumoral injection with M1 GAS wild-type, an isogenic mutant strain devoid of Scl1, or a complemented mutant strain with restored scl1 expression. In addition, recombinant Scl1 proteins were tested for NET inhibition using in vitro and ex vivo assays assessing NET production and myeloperoxidase activity. Results: Injection of three different M-type GAS strains reduced subcutaneous pancreatic tumor volume compared to control in two different murine PDAC models. Limitation of tumor growth was dependent on Scl1, as isogenic mutant strain devoid of Scl1 did not reduce tumor size. We further show that Scl1 plays a role in localizing GAS to the tumor site, thereby limiting the systemic spread of bacteria and off-target effects. While mice did elicit a humoral immune response to GAS antigens, tested sera were weakly immunogenic toward Scl1 antigen following intra-tumoral treatment with Scl1-expressing GAS. M1 GAS inhibited NET formation when co-cultured with neutrophils while Scl1-devoid mutant strain did not. Recombinant Scl1 protein inhibited NETs ex vivo in a dose-dependent manner by suppressing myeloperoxidase activity. Discussion: Altogether, we demonstrate that intra-tumoral GAS injections reduce PDAC growth, which is facilitated by Scl1, in part through inhibition of cancer promoting NETs. This work offers a novel strategy by which NETs can be targeted through Scl1 protein and potentiates its use as a cancer therapeutic.

Aspergillus collagen-like genes (acl): identification, sequence polymorphism, and assessment for PCR-based pathogen detection.

The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects.

Crystallization and preliminary X-ray crystallographic analysis of the variable domain of Scl2.3, a streptococcal collagen-like protein from invasive M3-type Streptococcus pyogenes.

Streptococcal collagen-like proteins (Scls) are widely expressed by the well recognized human pathogen Streptococcus pyogenes. These surface proteins contain a signature central collagen-like region and an amino-terminal globular domain, termed the variable domain, which is protruded away from the cell surface by the collagen-like domain. Despite their recognized importance in bacterial pathogenicity, no structural information is presently available on proteins of the Scl class. The variable domain of Scl2 from invasive M3-type S. pyogenes has successfully been crystallized using vapour-diffusion methods. The crystals diffracted to 1.5 Šresolution and belonged to space group H32, with unit-cell parameters a = 44.23, b = 44.23, c = 227.83 Å. The crystal structure was solved by single-wavelength anomalous dispersion using anomalous signal from a europium chloride derivative.|

Intranasal immunization with a Bucl8-based vaccine ameliorates bacterial burden and pathological inflammation, and promotes an IgG2a/b dominant response in an outbred mouse model of Burkholderia infection.

Burkholderia pseudomallei is a gram-negative bacterium that is the etiological agent of the tropical disease melioidosis. Currently, there is no licensed vaccine for melioidosis, but numerous candidates are being tested for protective efficacy and characterization of the elicited immune response. Our lab has previously reported the immunogenicity of a Bucl8-protein-based peptide antigen, designated L1-CRM197 (Cross-reacting material 197). When given subcutaneously, this vaccine formulation promoted a strong Th2 (IgG1) antibody response, however immunization did not protect from death. In this study, we hypothesized that an intranasally administered L1-CRM197 vaccine would induce protective mucosal immunity. To evaluate vaccine efficacy, we developed a surrogate Burkholderia infection model that employs outbred CD-1 mice which imitates the immunogenetic diversity of humans. Mice were immunized with either L1-CRM197 adjuvanted with fluorinated cyclic diguanosine monophosphate (FCDG) or with FCDG-only control. These mice were then challenged intranasally with an infectious dose of a luminescent strain of B. thailandensis E264 two weeks post-immunization, and correlates of protection were assessed in euthanized mice on days 1, 2, 3, and 7 post-infection. Overall, intranasal vaccination, compared to subcutaneous administration, induced a stronger Th1 (IgG2a/2b) to Th2 (IgG1) antibody response and promoted anti-L1 nasal, pulmonary, and systemic IgA. Additionally, sera IgG from L1-CRM197-vaccinated mice recognized whole-cell B. thailandensis and B. pseudomallei, a select agent exempt strain Bp82. Vaccination ameliorated disease indicators, including luminescent signal and bacterial cell counts, weight and temperature loss, and organ weight, which negatively correlated with IgG2a antibody levels and mucosa-stimulating cytokines IL-13 and IL-9. L1-CRM197-vaccinated mice also had earlier resolution of inflammatory and tissue-damaging cytokines compared to the FCDG-only controls. These results suggest a balanced humoral and cell-mediated response, along with mucosa-based immunity are beneficial for protection. Future efforts should further assess mucosal cellular and humoral mechanisms of protection and test such protection, using aerosolized B. pseudomallei select agent strain(s).

Emerging applications of cancer bacteriotherapy towards treatment of pancreatic cancer.

Pancreatic cancer is a highly aggressive form of cancer with a five-year survival rate of only ten percent. Pancreatic ductal adenocarcinoma (PDAC) accounts for ninety percent of those cases. PDAC is associated with a dense stroma that confers resistance to current treatment modalities. Increasing resistance to cancer treatments poses a challenge and a need for alternative therapies. Bacterial mediated cancer therapies were proposed in the late 1800s by Dr. William Coley when he injected osteosarcoma patients with live streptococci or a fabrication of heat-killed Streptococcus pyogenes and Serratia marcescens known as Coley's toxin. Since then, several bacteria have gained recognition for possible roles in potentiating treatment response, enhancing anti-tumor immunity, and alleviating adverse effects to standard treatment options. This review highlights key bacterial mechanisms and structures that promote anti-tumor immunity, challenges and risks associated with bacterial mediated cancer therapies, and applications and opportunities for use in PDAC management.

Binding of the human complement regulators CFHR1 and factor H by streptococcal collagen-like protein 1 (Scl1) via their conserved C termini allows control of the complement cascade at multiple levels.

Group A streptococci (GAS) utilize soluble human complement regulators to evade host complement attack. Here, we characterized the binding of the terminal complement complex inhibitor complement Factor H-related protein 1 (CFHR1) and of the C3 convertase regulator Factor H to the streptococcal collagen-like proteins (Scl). CFHR1 and Factor H, but no other member of the Factor H protein family (CFHR2, CFHR3, or CFHR4A), bound to the two streptococcal proteins Scl1.6 and Scl1.55, which are expressed by GAS serotypes M6 and M55. The two human regulators bound to the Scl1 proteins via their conserved C-terminal attachment region, i.e. CFHR1 short consensus repeats 3-5 (SCR3-5) and Factor H SCR18-20. Binding was affected by ionic strength and by heparin. CFHR1 and the C-terminal attachment region of Factor H did not bind to Scl1.1 and Scl2.28 proteins but did bind to intact M1-type and M28-type GAS, which express Scl1.1 and Scl2.28, respectively, thus arguing for the presence of an additional binding mechanism to CFHR1 and Factor H. Furthermore mutations within the C-terminal heparin-binding region and Factor H mutations that are associated with the acute renal disease atypical hemolytic uremic syndrome blocked the interaction with the two streptococcal proteins. Binding of CFHR1 affected the complement regulatory functions of Factor H on the level of the C3 convertase. Apparently, streptococci utilize two types of complement regulator-acquiring surface proteins; type A proteins, as represented by Scl1.6 and Scl1.55, bind to CFHR1 and Factor H via their conserved C-terminal region and do not bind the Factor H-like protein 1 (FHL-1). On the contrary, type B proteins, represented by M-, M-like, and the fibronectin-binding protein Fba proteins, bind Factor H and FHL-1 via domain SCR7 and do not bind CFHR1. In conclusion, binding of CFHR1 is at the expense of Factor H-mediated regulatory function at the level of C3 convertase and at the gain of a regulator that controls complement at the level of the C5 convertase and formation of the terminal complement complex.

The streptococcal collagen-like protein-1 (Scl1) is a significant determinant for biofilm formation by group A Streptococcus.

BACKGROUND: Group A Streptococcus (GAS) is a human-specific pathogen responsible for a number of diseases characterized by a wide range of clinical manifestations. During host colonization GAS-cell aggregates or microcolonies are observed in tissues. GAS biofilm, which is an in vitro equivalent of tissue microcolony, has only recently been studied and little is known about the specific surface determinants that aid biofilm formation. In this study, we demonstrate that surface-associated streptococcal collagen-like protein-1 (Scl1) plays an important role in GAS biofilm formation. RESULTS: Biofilm formation by M1-, M3-, M28-, and M41-type GAS strains, representing an intraspecies breadth, were analyzed spectrophotometrically following crystal violet staining, and characterized using confocal and field emission scanning electron microscopy. The M41-type strain formed the most robust biofilm under static conditions, followed by M28- and M1-type strains, while the M3-type strains analyzed here did not form biofilm under the same experimental conditions. Differences in architecture and cell-surface morphology were observed in biofilms formed by the M1- and M41-wild-type strains, accompanied by varying amounts of deposited extracellular matrix and differences in cell-to-cell junctions within each biofilm. Importantly, all Scl1-negative mutants examined showed significantly decreased ability to form biofilm in vitro. Furthermore, the Scl1 protein expressed on the surface of a heterologous host, Lactococcus lactis, was sufficient to induce biofilm formation by this organism. CONCLUSIONS: Overall, this work (i) identifies variations in biofilm formation capacity among pathogenically different GAS strains, (ii) identifies GAS surface properties that may aid in biofilm stability and, (iii) establishes that the Scl1 surface protein is an important determinant of GAS biofilm, which is sufficient to enable biofilm formation in the heterologous host Lactococcus. In summary, the GAS surface adhesin Scl1 may have an important role in biofilm-associated pathogenicity.

Characterization of recombinant terrelysin, a hemolysin of Aspergillus terreus.

Fungal hemolysins are potential virulence factors. Some fungal hemolysins belong to the aegerolysin protein family that includes cytolysins capable of lysing erythrocytes and other cells. Here, we describe a hemolysin from Aspergillus terreus called terrelysin. We used the genome sequence database to identify the terrelysin sequence based on homology with other known aegerolysins. Aspergillus terreus mRNA was isolated, transcribed to cDNA and the open reading frame for terrelysin amplified by PCR using specific primers. Using the pASK-IBA6 cloning vector, we produced recombinant terrelysin (rTerrelysin) as a fusion product in Escherichia coli. The recombinant protein was purified and using MALDI-TOF MS determined to have a mass of 16,428 Da. Circular dichroism analysis suggests the secondary structure of the protein to be predominantly ß-sheet. Results from thermal denaturation of rTerrelysin show that the protein maintained the ß-sheet confirmation up to 65°C. Polyclonal antibody to rTerrelysin recognized a protein of approximately 16.5 kDa in mycelial extracts from A. terreus.

Use of 16S ribosomal RNA gene analyses to characterize the bacterial signature associated with poor oral health in West Virginia.

BACKGROUND: West Virginia has the worst oral health in the United States, but the reasons for this are unclear. This pilot study explored the etiology of this disparity using culture-independent analyses to identify bacterial species associated with oral disease. METHODS: Bacteria in subgingival plaque samples from twelve participants in two independent West Virginia dental-related studies were characterized using 16S rRNA gene sequencing and Human Oral Microbe Identification Microarray (HOMIM) analysis. Unifrac analysis was used to characterize phylogenetic differences between bacterial communities obtained from plaque of participants with low or high oral disease, which was further evaluated using clustering and Principal Coordinate Analysis. RESULTS: Statistically different bacterial signatures (P<0.001) were identified in subgingival plaque of individuals with low or high oral disease in West Virginia based on 16S rRNA gene sequencing. Low disease contained a high frequency of Veillonella and Streptococcus, with a moderate number of Capnocytophaga. High disease exhibited substantially increased bacterial diversity and included a large proportion of Clostridiales cluster bacteria (Selenomonas, Eubacterium, Dialister). Phylogenetic trees constructed using 16S rRNA gene sequencing revealed that Clostridiales were repeated colonizers in plaque associated with high oral disease, providing evidence that the oral environment is somehow influencing the bacterial signature linked to disease. CONCLUSIONS: Culture-independent analyses identified an atypical bacterial signature associated with high oral disease in West Virginians and provided evidence that the oral environment influenced this signature. Both findings provide insight into the etiology of the oral disparity in West Virginia.

Immune Cell Modulation of the Extracellular Matrix Contributes to the Pathogenesis of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with a five-year survival rate of only 9%. PDAC is characterized by a dense, fibrotic stroma composed of extracellular matrix (ECM) proteins. This desmoplastic stroma is a hallmark of PDAC, representing a significant physical barrier that is immunosuppressive and obstructs penetration of cytotoxic chemotherapy agents into the tumor microenvironment (TME). Additionally, dense ECM promotes hypoxia, making tumor cells refractive to radiation therapy and alters their metabolism, thereby supporting proliferation and survival. In this review, we outline the significant contribution of fibrosis to the pathogenesis of pancreatic cancer, with a focus on the cross talk between immune cells and pancreatic stellate cells that contribute to ECM deposition. We emphasize the cellular mechanisms by which neutrophils and macrophages, specifically, modulate the ECM in favor of PDAC-progression. Furthermore, we investigate how activated stellate cells and ECM influence immune cells and promote immunosuppression in PDAC. Finally, we summarize therapeutic strategies that target the stroma and hinder immune cell promotion of fibrogenesis, which have unfortunately led to mixed results. An enhanced understanding of the complex interactions between the pancreatic tumor ECM and immune cells may uncover novel treatment strategies that are desperately needed for this devastating disease.