The role of O-polysaccharide chain and complement resistance of Escherichia coli in mammary virulence.

Mastitis, inflammation of the mammary gland, is a common disease of dairy animals. The disease is caused by bacterial infection ascending through the teat canal and mammary pathogenic Escherichia coli (MPEC) are common etiology. In the first phase of infection, virulence mechanisms, designated as niche factors, enable MPEC bacteria to resist innate antimicrobial mechanisms, replicate in milk, and to colonize the mammary gland. Next, massive replication of colonizing bacteria culminates in a large biomass of microbe-associated molecular patterns (MAMPs) recognized by pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) mediating inflammatory signaling in mammary alveolar epithelial cells (MAEs) and macrophages. Bacterial lipopolysaccharides (LPSs), the prototypical class of MAMPs are sufficient to elicit mammary inflammation mediated by TLR4 signaling and activation of nuclear factor kB (NF-kB), the master regulator of inflammation. Using in vivo mastitis model, in low and high complements mice, and in vitro NF-kB luminescence reporter system in MAEs, we have found that the smooth configuration of LPS O-polysaccharides in MPEC enables the colonizing organisms to evade the host immune response by reducing inflammatory response and conferring resistance to complement. Screening a collection of MPEC field strains, we also found that all strains were complement resistant and 94% (45/48) were smooth. These results indicate that the structure of LPS O-polysaccharides chain is important for the pathogenesis of MPEC mastitis and provides protection against complement-mediated killing. Furthermore, we demonstrate a role for complement, a key component of innate immunity, in host-microbe interactions of the mammary gland.

Intraepithelial neutrophils in mammary, urinary and gall bladder infections.

Neutrophil mobilization is a crucial response to protect the host against invading microorganisms. Neutrophil recruitment and removal have to be tightly regulated to prevent uncontrolled inflammation and excessive release of their toxic content causing tissue damage and subsequent organ dysfunctions. We show here the presence of live and apoptotic neutrophils in the cytoplasm of inflamed mammary, urinary and gall bladder epithelial cells following infection with E. coli and Salmonella bacteria. The entry process commenced with adherence of transmigrated neutrophils to the apical membrane of inflamed epithelial cells. Next, nuclear rearrangement and elongation associated with extensive actin polymerization enabled neutrophils to crawl and invaginate the apical membrane into cytoplasmic double membrane compartments. Scission of the invaginated cell membrane from the entry point and loss of these surrounding membranes released intracellular neutrophils into the cytoplasm where they undergone apoptotic death. The co-occurrence of this observation with bacterial invasion and formation of intracellular bacterial communities (IBCs) might link entry of infected neutrophils to the formation of IBCs and chronic carriage in E. coli mastitis and cystitis and Salmonella cholecystitis.

Immune profiling of experimental murine mastitis reveals conserved response to mammary pathogenic Escherichia coli, Mycoplasma bovis, and Streptococcus uberis.

Mastitis is one of the most prevalent and economically important diseases of dairy animals. The disease is caused by ascending bacterial infection through the teat canal. Among the most common mastitis-causing bacteria are Gram-negative coliforms, Gram-positive streptococci and staphylococci, and mycoplasma. The most prominent cellular hallmark of acute mammary infection is a massive recruitment of blood neutrophils into the tubular and alveolar milk spaces. The complex biological processes of leukocyte recruitment, activation, adhesion, and migration in the mammary gland remain largely elusive to date. While field research of mastitis in dairy animals contributed a lot to the development of mitigation, control, and even eradication programs, little progress was made toward understanding the molecular mechanisms underlying the pathogenesis of the disease. We report here experimental mastitis model systems in lactating mice challenged with field strains of common udder pathogens in dairy cows. We used these model systems to apply recently developed multiplex gene expression technology (Nanostring nCounter), which enabled us to study the expression of over 700 immune genes. Our analysis revealed a core of 100 genes that are similarly regulated and functionally or physically interacting in E. coli, M. bovis, and Strep uberis murine mastitis. Common significantly enriched gene sets include TNFɑ signaling via NFkB, Interferon gamma and alpha response, and IL6-JAK-STAT3 signaling. In addition, we show a significantly enriched expression of genes associated with neutrophil extracellular traps (NET) in glands challenged by the three pathogens. Ligand-receptor analysis revealed interactions shared by the three pathogens, including the interaction of the cytokines IL1ß, IL1ɑ, and TNFɑ with their receptors, and proteins involved in immune cell recruitment such as complement C3 and ICAM1 (with CD11b), chemokines CCL3 and CCL4 (with CCR1), and CSF3 (with CSF3R). Taken together, our results show that mammary infection with E. coli, M. bovis, and Strep uberis culminated in the activation of a conserved core of immune genes and pathways including NET formation.