Staphylococcus Aureus Biofilms Interfere With Macrophage Antimicrobial Responses Through Differential Gene Regulation, Toxin Production, and Purine Metabolism

Staphylococcus aureus (S. aureus) is an opportunistic pathogen that is a leading cause of both nosocomial and community-associated infections. Armed with a myriad of virulence factors and the propensity to form a biofilm on native tissues and implanted medical devices alike, S. aureus infections represent a very real public health threat, the treatment of which results in an excessive economic burden. S. aureus biofilm infections are notoriously recalcitrant to antibiotic therapy and adept at evading and neutralizing the host immune antimicrobial response. Previous studies from our laboratory have shown that S. aureus biofilms are able to cause persistent infections, in part, through the reprogramming of the macrophage (MΦ) immune response. While macrophages are readily able to recognize and respond to S. aureus in a planktonic state, their ability to mount a functional antimicrobial attack is thwarted upon encountering S. aureus biofilm. We have observed that MΦs in close proximity to S. aureus biofilms are less phagocytic and skewed towards an anti-inflammatory profile typified by arginase and IL-10 production. We have demonstrated that the ability of S. aureus biofilms to cause chronic infections is due, in part, to TLR2 or TLR9 evasion. However, we have shown that MyD88 signaling does provide some benefit to the host in combating S. aureus biofilm infections, which may be attributed to IL-1 receptor signaling. To better understand how S. aureus biofilms subvert the MΦ antimicrobial response, the work described in this dissertation assessed S. aureus transcriptional activity during co-culture with MΦs, whether S. aureus biofilms inhibit MΦ activity through secreted molecules, and performed a high-throughput screen of the Nebraska Transposon Mutant Library to identify key genes involved in dampening the MΦ NF-κB-regulated proinflammatory response. We found that S. aureus biofilms attenuate their transcriptional activity following MΦ exposure, augment α-hemolysin (Hla) and leukocidin AB (LukAB) secretion to inhibit MΦ phagocytosis and induce cell death, and rely on a functional purine biosynthetic pathway to prevent MΦ invasion and phagocytosis, in part, through controlling the amount of eDNA available for MΦ recognition at the surface of the biofilm extracellular matrix (ECM). Collectively, these studies build upon our previous observations by identifying key mechanisms whereby S. aureus biofilms are able to thwart the MΦ antimicrobial response

Hiding in Plain Sight: Interplay between Staphylococcal Biofilms and Host Immunity.

Staphylococcus aureus and Staphylococcus epidermidis are notable for their propensity to form biofilms on implanted medical devices. Staphylococcal biofilm infections are typified by their recalcitrance to antibiotics and ability to circumvent host immune-mediated clearance, resulting in the establishment of chronic infections that are often recurrent in nature. Indeed, the immunomodulatory lifestyle of biofilms seemingly shapes the host immune response to ensure biofilm engraftment and persistence in an immune competent host. Here, we provide a brief review of the mechanisms whereby S. aureus and S. epidermidis biofilms manipulate host-pathogen interactions and discuss the concept of microenvironment maintenance in infectious outcomes, as well as speculate how these findings pertain to the challenges of staphylococcal vaccine development

Staphylococcus aureus Biofilms Induce Macrophage Dysfunction Through Leukocidin AB and Alpha-Toxin.

UNLABELLED: The macrophage response to planktonic Staphylococcus aureus involves the induction of proinflammatory microbicidal activity. However, S. aureus biofilms can interfere with these responses in part by polarizing macrophages toward an anti-inflammatory profibrotic phenotype. Here we demonstrate that conditioned medium from mature S. aureus biofilms inhibited macrophage phagocytosis and induced cytotoxicity, suggesting the involvement of a secreted factor(s). Iterative testing found the active factor(s) to be proteinaceous and partially agr-dependent. Quantitative mass spectrometry identified alpha-toxin (Hla) and leukocidin AB (LukAB) as critical molecules secreted by S. aureus biofilms that inhibit murine macrophage phagocytosis and promote cytotoxicity. A role for Hla and LukAB was confirmed by using hla and lukAB mutants, and synergy between the two toxins was demonstrated with a lukAB hla double mutant and verified by complementation. Independent confirmation of the effects of Hla and LukAB on macrophage dysfunction was demonstrated by using an isogenic strain in which Hla was constitutively expressed, an Hla antibody to block toxin activity, and purified LukAB peptide. The importance of Hla and LukAB during S. aureus biofilm formation in vivo was assessed by using a murine orthopedic implant biofilm infection model in which the lukAB hla double mutant displayed significantly lower bacterial burdens and more macrophage infiltrates than each single mutant. Collectively, these findings reveal a critical synergistic role for Hla and LukAB in promoting macrophage dysfunction and facilitating S. aureus biofilm development in vivo. IMPORTANCE: Staphylococcus aureus has a propensity to form multicellular communities known as biofilms. While growing in a biofilm, S. aureus displays increased tolerance to nutrient deprivation, antibiotic insult, and even host immune challenge. Previous studies have shown that S. aureus biofilms thwart host immunity in part by preventing macrophage phagocytosis. It remained unclear whether this was influenced solely by the considerable size of biofilms or whether molecules were also actively secreted to circumvent macrophage-mediated phagocytosis. This is the first report to demonstrate that S. aureus biofilms inhibit macrophage phagocytosis and induce macrophage death through the combined action of leukocidin AB and alpha-toxin. Loss of leukocidin AB and alpha-toxin expression resulted in enhanced S. aureus biofilm clearance in a mouse model of orthopedic implant infection, suggesting that these toxins could be targeted therapeutically to facilitate biofilm clearance in humans

Upper tract urothelial carcinoma has a luminal-papillary T-cell depleted contexture and activated FGFR3 signaling.

Upper tract urothelial carcinoma (UTUC) is characterized by a distinctly aggressive clinical phenotype. To define the biological features driving this phenotype, we performed an integrated analysis of whole-exome and RNA sequencing of UTUC. Here we report several key insights from our molecular dissection of this disease: 1) Most UTUCs are luminal-papillary; 2) UTUC has a T-cell depleted immune contexture; 3) High FGFR3 expression is enriched in UTUC and correlates with its T-cell depleted immune microenvironment; 4) Sporadic UTUC is characterized by a lower total mutational burden than urothelial carcinoma of the bladder. Our findings lay the foundation for a deeper understanding of UTUC biology and provide a rationale for the development of UTUC-specific treatment strategies