Skin provides the first defense against pathogenic micro-organisms and is also colonized by a diverse microbiota. Phylogenetic analysis of whole skin microbiome at different skin sites in health and disease has generated important insights on possible microbial involvement in modulating skin health. However, functional roles of the skin microbial community remain unclear. The most common sebaceous skin commensal yeasts are the basidiomycetes, Malassezia. Here, we characterized the dominant secreted Malassezia globosa protease in culture and subsequently named it Malassezia globosa Secreted Aspartyl Protease 1 (MgSAP1). We defined recombinant MgSAP1's substrate cleavage profile using an unbiased, mass-spectrometry-based technique. We show that this enzyme is physiologically relevant as mgsap1 expression was detected on at least one facial skin site of 17 healthy human volunteers. In addition, we demonstrated that this protease rapidly hydrolyzes Staphylococcus aureus protein A, an important S. aureus virulence factor involved in immune evasion and biofilm formation. We further observed that MgSAP1 has anti-biofilm properties against S. aureus. Taken together, our study defines a role for the skin fungus Malassezia in inter-kingdom interactions and suggests that this fungus and the enzymes it produces may be beneficial for skin health.

Cyclic di-AMP is a recently identified second messenger exploited by a number of Gram-positive bacteria to regulate important biological processes. Here, we studied the phenotypic alterations induced by the increased intracellular c-di-AMP levels in Streptococcus gallolyticus, an opportunistic pathogen responsible for septicemia and endocarditis in the elderly. We report that an S. gallolyticus c-di-AMP phosphodiesterase gdpP knockout mutant, which displays a 1.5-fold higher intracellular c-di-AMP levels than the parental strain UCN34, is more sensitive to osmotic stress and is morphologically smaller than the parental strain. Unexpectedly, we found that a higher level of c-di-AMP reduced biofilm formation of S. gallolyticus on abiotic surfaces and reduced adherence and cell aggregation on human intestinal cells. A genome-wide transcriptomic analysis indicated that c-di-AMP regulates many biological processes in S. gallolyticus, including the expression of various ABC transporters and disease-associated genes encoding bacteriocin and Pil3 pilus. Complementation of the gdpP in-frame deletion mutant with a plasmid carrying gdpP in trans from its native promoter restored bacterial morphology, tolerance to osmotic stress, biofilm formation, adherence to intestinal cells, bacteriocin production, and Pil3 pilus expression. Our results indicate that c-di-AMP is a pleiotropic signaling molecule in S. gallolyticus that may be important for S. gallolyticus pathogenesis. IMPORTANCE Streptococcus gallolyticus is an opportunistic pathogen responsible for septicemia and endocarditis in the elderly and is also strongly associated with colorectal cancer. S. gallolyticus can form biofilms, express specific pili to colonize the host tissues, and produce a specific bacteriocin allowing killing of commensal bacteria in the murine colon. Nevertheless, how the expression of these colonization factors is regulated remains largely unknown. Here, we show that c-di-AMP plays pleiotropic roles in S. gallolyticus, controlling the tolerance to osmotic stress, cell size, biofilm formation on abiotic surfaces, adherence and cell aggregation on human intestinal cells, expression of Pil3 pilus, and production of bacteriocin. This study indicates that c-di-AMP may constitute a key regulatory molecule for S. gallolyticus host colonization and pathogenesis.

Secondary bacterial lung infection by Streptococcus pneumoniae (S. pneumoniae) poses a serious health concern, especially in developing countries. We posit that the emergence of multiantibiotic-resistant strains will jeopardize current treatments in these regions. Deaths arising from secondary infections are more often associated with acute lung injury, a common consequence of hypercytokinemia, than with the infection per se. Given that secondary bacterial pneumonia often has a poor prognosis, newer approaches to improve treatment outcomes are urgently needed to reduce the high levels of morbidity and mortality. Using a sequential dual-infection mouse model of secondary bacterial lung infection, we show that host-directed therapy via immunoneutralization of the angiopoietin-like 4 c-isoform (cANGPTL4) reduced pulmonary edema and damage in infected mice. RNA sequencing analysis revealed that anti-cANGPTL4 treatment improved immune and coagulation functions and reduced internal bleeding and edema. Importantly, anti-cANGPTL4 antibody, when used concurrently with either conventional antibiotics or antipneumolysin antibody, prolonged the median survival of mice compared to monotherapy. Anti-cANGPTL4 treatment enhanced immune cell phagocytosis of bacteria while restricting excessive inflammation. This modification of immune responses improved the disease outcomes of secondary pneumococcal pneumonia. Taken together, our study emphasizes that host-directed therapeutic strategies are viable adjuncts to standard antimicrobial treatments. IMPORTANCE Despite extensive global efforts, secondary bacterial pneumonia still represents a major cause of death in developing countries and is an important cause of long-term functional disability arising from lung tissue damage. Newer approaches to improving treatment outcomes are needed to reduce the significant morbidity and mortality caused by infectious diseases. Our study, using an experimental mouse model of secondary S. pneumoniae infection, shows that a multimodal treatment that concurrently targets host and pathogen factors improved lung tissue integrity and extended the median survival time of infected mice. The immunoneutralization of host protein cANGPTL4 reduced the severity of pulmonary edema and damage. We show that host-directed therapeutic strategies as well as neutralizing antibodies against pathogen virulence factors are viable adjuncts to standard antimicrobial treatments such as antibiotics. In view of their different modes of action compared to antibiotics, concurrent immunotherapies using antibodies are potentially efficacious against secondary pneumococcal pneumonia caused by antibiotic-resistant pathogens.

Biofilms are surface-attached communities of microorganisms embedded in an extracellular matrix and are essential for the cycling of organic matter in natural and engineered environments. They are also the leading cause of many infections, for example, those associated with chronic wounds and implanted medical devices. The extracellular matrix is a key biofilm component that determines its architecture and defines its physical properties. Herein, we used growth chambers embedded with micropillars to study the net mechanical forces (differential pressure) exerted during biofilm formation in situ. Pressure from the biofilm is transferred to the micropillars via the extracellular matrix, and reduction of major matrix components decreases the magnitude of micropillar deflections. The spatial arrangement of micropillar deflections caused by pressure differences in the different biofilm strains may potentially be used as mechanical signatures for biofilm characterization. Hence, we submit that micropillar-embedded growth chambers provide insights into the mechanical properties and dynamics of the biofilm and its matrix.

Streptococcus gallolyticus subsp. gallolyticus (Sgg) is an opportunistic bacterial pathogen strongly associated with colorectal cancer. Here, through comparative genomics analysis, we demonstrated that the genetic locus encoding the Type VIIb Secretion System (T7SSb) machinery is uniquely present in Sgg in two different arrangements. Sgg UCN34 carrying the most prevalent T7SSb genetic arrangement was chosen as the reference strain. To identify the effectors secreted by T7SSb, we inactivated the essC gene encoding the motor of this machinery. Comparison of the proteins secreted by Sgg UCN34 WT and its isogenic ∆essC mutant revealed six T7SSb effector proteins, including an LXG-family toxin named herein TelD displaying pore-forming activity. Seven homologs of TelD harboring a conserved glycine zipper motif at the C-terminus were identified in Sgg isolates. Scanning mutagenesis of this motif showed that the glycine residue at position 470 was crucial for TelD pore-forming activity. Unlike other pore-forming toxins commonly antagonized by a membrane protein, TelD activity was antagonized by a cytosolic protein TipD belonging to the DUF5085 family. Finally, we demonstrated that purified TelD can permeabilize E. coli cells rapidly when introduced extracellularly. Overall, we report herein a unique Sgg T7SSb effector exhibiting a pore-forming activity against non-immune bacteria.

In this study, 38 clinical isolates of Streptococcus gallolyticus subsp. gallolyticus ( SGG ) were sequenced and a genetic locus encoding the type VIIb secretion system (T7SSb) was found conserved and absent from 16 genomes of the closely related S. gallolyticus subsp. pasteurianus ( SGP ). The T7SSb is a bona fide pathogenicity island.

Chapter 1 -Introduction 1.1 Background information Streptococcus gallolyticus subsp. gallolyticus, a Lancefield Group D Streptococcus, is an emerging opportunistic pathogen. It is one of the gut microbiota members present in 2.5 to 15% of healthy individuals (1). Upon entering into bloodstream, S. gallolyticus subsp. gallolyticus can colonise damaged heart valves, causing infective endocarditis. S. gallolyticus subsp. gallolyticus infections can represent up to 25% of all infective endocarditis cases (2). S. gallolyticus subsp. gallolyticus was previously known as S. bovis biotype I, a traditional nomenclature that has been replaced with a system that clearly differentiate S. gallolyticus at the subspecies level. The renaming of S. bovis is clinically important. Unlike S. infantarius subsp. coli (biotype II.1) and S. gallolyticus subsp. pasteurianus (biotype II.2), S. gallolyticus subsp. gallolyticus is the specific S. gallolyticus that exhibits a strong association with the incidence of colorectal cancer among the endocarditis patients infected with S. gallolyticus (3-6). The significant enrichment of S. gallolyticus subsp. gallolyticus in the stools of these patients was reported as early as in 1951, and subsequently supported by many case reports and systemic reviews (1, 6-10). Due to the strong association of S. gallolyticus subsp. gallolyticus with colorectal cancer, UptoDate database currently lists a follow up colonoscopy as a strong medical recommendation for the endocarditis patients infected with S. gallolyticus.