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.

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.

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.

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.

Antibody responses to collagen peptides and streptococcal collagen-like 1 proteins in acute rheumatic fever patients.

Acute rheumatic fever (ARF) is a serious post-infectious immune sequelae of Group A streptococcus (GAS). Pathogenesis remains poorly understood, including the events associated with collagen autoantibody generation. GAS express streptococcal collagen-like proteins (Scl) that contain a collagenous domain resembling human collagen. Here, the relationship between antibody reactivity to GAS Scl proteins and human collagen in ARF was investigated. Serum IgG specific for a representative Scl protein (Scl1.1) together with collagen-I and collagen-IV mimetic peptides were quantified in ARF patients (n = 36) and healthy matched controls (n = 36). Reactivity to Scl1.1 was significantly elevated in ARF compared to controls (P < 0.0001) and this was mapped to the collagen-like region of the protein, rather than the N-terminal non-collagenous region. Reactivity to collagen-1 and collagen-IV peptides was also significantly elevated in ARF cases (P < 0.001). However, there was no correlation between Scl1.1 and collagen peptide antibody binding, and hierarchical clustering of ARF cases by IgG reactivity showed two distinct clusters, with Scl1.1 antigens in one and collagen peptides in the other, demonstrating that collagen autoantibodies are not immunologically related to those targeting Scl1.1. Thus, anti-collagen antibodies in ARF appear to be generated as part of the autoreactivity process, independent of any mimicry with GAS collagen-like proteins.

Thinking Outside the Bug: Targeting Outer Membrane Proteins for Burkholderia Vaccines.

Increasing antimicrobial resistance due to misuse and overuse of antimicrobials, as well as a lack of new and innovative antibiotics in development has become an alarming global threat. Preventative therapeutics, like vaccines, are combative measures that aim to stop infections at the source, thereby decreasing the overall use of antibiotics. Infections due to Gram-negative pathogens pose a significant treatment challenge because of substantial multidrug resistance that is acquired and spread throughout the bacterial population. Burkholderia spp. are Gram-negative intrinsically resistant bacteria that are responsible for environmental and nosocomial infections. The Burkholderia cepacia complex are respiratory pathogens that primarily infect immunocompromised and cystic fibrosis patients, and are acquired through contaminated products and equipment, or via patient-to-patient transmission. The Burkholderia pseudomallei complex causes percutaneous wound, cardiovascular, and respiratory infections. Transmission occurs through direct exposure to contaminated water, water-vapors, or soil, leading to the human disease melioidosis, or the equine disease glanders. Currently there is no licensed vaccine against any Burkholderia pathogen. This review will discuss Burkholderia vaccine candidates derived from outer membrane proteins, OmpA, OmpW, Omp85, and Bucl8, encompassing their structures, conservation, and vaccine formulation.

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.

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.

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.

Biofilm formation by group A Streptococcus: a role for the streptococcal regulator of virulence (Srv) and streptococcal cysteine protease (SpeB).

Recently, biofilms have become a topic of interest in the study of the human pathogen group A Streptococcus (GAS). In this study, we sought to learn more about the make-up of these structures and gain insight into biofilm regulation. Enzymic studies indicated that biofilm formation by GAS strain MGAS5005 required an extracellular protein and DNA component(s). Previous results indicated that inactivation of the transcriptional regulator Srv in MGAS5005 resulted in a significant decrease in virulence. Here, inactivation of Srv also resulted in a significant decrease in biofilm formation under both static and flow conditions. Given that production of the extracellular cysteine protease SpeB is increased in the srv mutant, we tested the hypothesis that increased levels of active SpeB may be responsible for the reduction in biofilm formation. Western immunoblot analysis indicated that SpeB was absent from MGAS5005 biofilms. Complementation of MGAS5005Deltasrv restored the biofilm phenotype and eliminated the overproduction of active SpeB. Inhibition of SpeB with E64 also restored the MGAS5005Deltasrv biofilm to wild-type levels.

Scl1-dependent internalization of group A Streptococcus via direct interactions with the alpha2beta(1) integrin enhances pathogen survival and re-emergence.

The molecular pathogenesis of infections caused by group A Streptococcus (GAS) is not fully understood. We recently reported that a recombinant protein derived from the collagen-like surface protein, Scl1, bound to the human collagen receptor, integrin alpha(2)beta(1). Here, we investigate whether the same Scl1 variant expressed by GAS cells interacts with the integrin alpha2beta(1) and affects the biological outcome of host-pathogen interactions. We demonstrate that GAS adherence and internalization involve direct interactions between surface expressed Scl1 and the alpha2beta(1) integrin, because (i) both adherence and internalization of the scl1-inactivated mutant were significantly decreased, and were restored by in-trans complementation of Scl1 expression, (ii) GAS internalization was reduced by pre-treatment of HEp-2 cells with anti-alpha2 integrin-subunit antibody and type I collagen, (iii) recombinant alpha2-I domain bound the wild-type GAS cells and (iv) internalization of wild-type cells was significantly increased in C2C12 cells expressing the alpha2beta(1) integrin as the only collagen-binding integrin. Next, we determined that internalized GAS re-emerges from epithelial cells into the extracellular environment. Taken together, our data describe a new molecular mechanism used by GAS involving the direct interaction between Scl1 and integrins, which increases the overall capability of the pathogen to survive and re-emerge.

Assessment of prokaryotic collagen-like sequences derived from streptococcal Scl1 and Scl2 proteins as a source of recombinant GXY polymers.

Collagen triple helix, composed of the repeating Gly-Xaa-Yaa (GXY) sequence, is a structural element found in all multicellular animals and also in some prokaryotes. Long GXY polymers are highly regarded components used in food, cosmetic, biomedical, and pharmaceutical industries. In this study, we explore a new concept for the production of recombinant GXY polymers which are based on the sequence of "prokaryotic collagens", the streptococcal collagen-like proteins Scl1 and Scl2. Analysis of 50 Scl variants identified the amino acid distribution and GXY-repeat usage that are involved in the stabilization of the triple helix in Scls. Using circular dichroism spectroscopy and electron microscopy, we show that significantly different recombinant rScl polypeptides form stable, unhydroxylated homotrimeric triple helices that can be produced both intra- and extracellularly in the Escherichia coli. These rScl constructs containing 20 to 129 GXY repeats had mid-point melting temperatures between 32 and 39 degrees C. Altogether, Scl-derived collagens, which are different from the mammalian collagens, can form stable triple helices under physiological conditions and can be used for the production of recombinant GXY polymers with a wide variety of potential applications.

A streptococcal collagen-like protein interacts with the alpha2beta1 integrin and induces intracellular signaling.

The streptococcal collagen-like proteins Scl1 and Scl2 are prokaryotic members of a large protein family with domains containing the repeating amino acid sequence (Gly-Xaa-Yaa)(n) that form a collagen-like triple-helical structure. Here, we test the hypothesis that Scl variant might interact with mammalian collagen-binding integrins. We show that the recombinant Scl protein p176 promotes adhesion and spreading of human lung fibroblast cells through an alpha2beta1 integrin-mediated interaction as shown in cell adhesion inhibition assays using anti-alpha2beta1 and anti-beta1 integrins monoclonal antibodies. Accordingly, C2C12 cells stably expressing alpha2beta1 integrin as the only collagen-binding integrin show productive cell adhesion activities on p176 that can be blocked by an anti-alpha2beta1 integrin antibody. In addition, p176 promotes tyrosine phosphorylation of p125(FAK) of C2C12 cells expressing alpha2beta1 integrin, whereas parental cells do not. Furthermore, C2C12 adhesion of human lung fibroblast cells to p176 induces phosphorylation of p125FAK, p130CAS, and p68Paxillin proteins. In a domain swapping experiment, we show that integrin binds to the collagenous domain of the Scl protein. Moreover, the recombinant inserted domain of the alpha2 integrin interacts with p176 with a relatively high affinity (K(D) = 17 nm). Attempts to identify the integrin sites in p176 suggest that more than one site may be involved. These studies, for the first time, suggest that the collagen-like proteins of prokaryotes retained not only structural but also functional characteristics of their eukaryotic counterparts.