Host-pathogen interactions during Campylobacter and Yersinia infections

The innate immune system is known for protecting the host against invading pathogens, for instance enteropathogens infecting the gastrointestinal tract. The production of e.g. antimicrobial peptides, cytokines, and chemokines by innate immune cells and intestinal epithelial cells contribute to bacterial clearance. Given the significance of this system in overall defense, pathogens affect and/or manipulate immune cells and responses in favor of their own survival. This thesis focuses on how the Gram-negative enteropathogenic bacteria Yersinia pseudotuberculosis and Campylobacter jejuni affect the host, either directly via type 3 secretion system (T3SS) effector proteins or via outer membrane vesicles (OMVs), and how host factors potentially affect their virulence. Yersinia pseudotuberculosis uses its T3SS to translocate virulence factors that disable various immune responses and subvert phagocytosis. Neutrophils are main target cells during Yersinia infection. They release granules that contain proteins with antimicrobial properties to the cell's exterior upon activation through a process called degranulation. We found that extracellular Y. pseudotuberculosis could prevent neutrophil degranulation upon cell contact. Prevention of degranulation was shown to be mediated via co-operative actions of the two anti-phagocytic Yersinia outer proteins YopH and YopE. Bacterial contact with neutrophils resulted in a transient inhibition of degranulation and further prevented degranulation upon subsequent contact with avirulent Y. pseudotuberculosis (lacking YopE and YopH) as well as Escherichia coli. Thus, Y. pseudotuberculosis impairs several neutrophil defense mechanisms to remain in the extracellular environment and to increase its survival during infection. Campylobacter jejuni lacks a T3SS and appears to use OMVs and flagella as its main secretion apparatus. During passage through the intestine C. jejuni is exposed to bile, an important physiological component and part of the natural barrier of the intestine, and ability to resist bile is advantageous for C. jejuni survival. We investigated how C. jejuni OMV production and protein content is affected by bile. The main invasion and colonization of C. jejuni occurs in the lower part of the intestine where the concentration of bile is low compared with the proximal intestine. The OMV proteomic profiles were radically altered when bacteria were grown in low concentration of bile corresponding to cecal concentrations. Twenty-five present of the detected proteins of OMVs showed an altered abundance in the presence of low concentration of bile. In contrast, the overall proteome of the bacteria was unaffected. Moreover, OMVs frombile-exposed bacteria could enhance adhesion as well as invasion of bacteria into intestinal epithelial cells, suggesting a role of OMVs to the virulence of C. jejuni in the gut. The body temperature differs between the asymptomatic avian carriers of C. jejuni and humans, which develop symptomatic disease. We investigated whether the bacterial growth temperature affects the OMV proteome and found that 59 proteins were differentially expressed at 37°C. Among the higher abundant proteins, significantly more proteins were predicted to be related to virulence. Thus, temperature has an impact on the property of the OMVs, and this might affect the outcome of infection by C. jejuni in different hosts. C. jejuni OMV interactions with innate immune cells were studied by analyses of OMV-mediated inflammasome activation. OMVs were found to induce ASC- and caspase-1-dependent inflammasome activation in murine and human macrophages and dendritic cells as well as in human neutrophils. While C. jejuni infection induced a low level of inflammasome-dependent cell death, OMV-induced inflammasome activation did not result in cell death. Thus, OMVs disseminate into tissue without bacteria can be a vehicle for virulence factors without inducing inflammatory cell death

Host-pathogen interactions during Campylobacter and Yersinia infections

The innate immune system is known for protecting the host against invading pathogens, for instance enteropathogens infecting the gastrointestinal tract. The production of e.g. antimicrobial peptides, cytokines, and chemokines by innate immune cells and intestinal epithelial cells contribute to bacterial clearance. Given the significance of this system in overall defense, pathogens affect and/or manipulate immune cells and responses in favor of their own survival. This thesis focuses on how the Gram-negative enteropathogenic bacteria Yersinia pseudotuberculosis and Campylobacter jejuni affect the host, either directly via type 3 secretion system (T3SS) effector proteins or via outer membrane vesicles (OMVs), and how host factors potentially affect their virulence. Yersinia pseudotuberculosis uses its T3SS to translocate virulence factors that disable various immune responses and subvert phagocytosis. Neutrophils are main target cells during Yersinia infection. They release granules that contain proteins with antimicrobial properties to the cell's exterior upon activation through a process called degranulation. We found that extracellular Y. pseudotuberculosis could prevent neutrophil degranulation upon cell contact. Prevention of degranulation was shown to be mediated via co-operative actions of the two anti-phagocytic Yersinia outer proteins YopH and YopE. Bacterial contact with neutrophils resulted in a transient inhibition of degranulation and further prevented degranulation upon subsequent contact with avirulent Y. pseudotuberculosis (lacking YopE and YopH) as well as Escherichia coli. Thus, Y. pseudotuberculosis impairs several neutrophil defense mechanisms to remain in the extracellular environment and to increase its survival during infection. Campylobacter jejuni lacks a T3SS and appears to use OMVs and flagella as its main secretion apparatus. During passage through the intestine C. jejuni is exposed to bile, an important physiological component and part of the natural barrier of the intestine, and ability to resist bile is advantageous for C. jejuni survival. We investigated how C. jejuni OMV production and protein content is affected by bile. The main invasion and colonization of C. jejuni occurs in the lower part of the intestine where the concentration of bile is low compared with the proximal intestine. The OMV proteomic profiles were radically altered when bacteria were grown in low concentration of bile corresponding to cecal concentrations. Twenty-five present of the detected proteins of OMVs showed an altered abundance in the presence of low concentration of bile. In contrast, the overall proteome of the bacteria was unaffected. Moreover, OMVs frombile-exposed bacteria could enhance adhesion as well as invasion of bacteria into intestinal epithelial cells, suggesting a role of OMVs to the virulence of C. jejuni in the gut. The body temperature differs between the asymptomatic avian carriers of C. jejuni and humans, which develop symptomatic disease. We investigated whether the bacterial growth temperature affects the OMV proteome and found that 59 proteins were differentially expressed at 37°C. Among the higher abundant proteins, significantly more proteins were predicted to be related to virulence. Thus, temperature has an impact on the property of the OMVs, and this might affect the outcome of infection by C. jejuni in different hosts. C. jejuni OMV interactions with innate immune cells were studied by analyses of OMV-mediated inflammasome activation. OMVs were found to induce ASC- and caspase-1-dependent inflammasome activation in murine and human macrophages and dendritic cells as well as in human neutrophils. While C. jejuni infection induced a low level of inflammasome-dependent cell death, OMV-induced inflammasome activation did not result in cell death. Thus, OMVs disseminate into tissue without bacteria can be a vehicle for virulence factors without inducing inflammatory cell death

Diversity in genetic regulation of bacterial fimbriae assembled by the chaperone usher pathway

Bacteria express different types of hair-like proteinaceous appendages on their cell surface known as pili or fimbriae. These filamentous structures are primarily involved in the adherence of bacteria to both abiotic and biotic surfaces for biofilm formation and/or virulence of non-pathogenic and pathogenic bacteria. In pathogenic bacteria, especially Gram-negative bacteria, fimbriae play a key role in bacteria–host interactions which are critical for bacterial invasion and infection. Fimbriae assembled by the Chaperone Usher pathway (CUP) are widespread within the Enterobacteriaceae, and their expression is tightly regulated by specific environmental stimuli. Genes essential for expression of CUP fimbriae are organised in small blocks/clusters, which are often located in proximity to other virulence genes on a pathogenicity island. Since these surface appendages play a crucial role in bacterial virulence, they have potential to be harnessed in vaccine development. This review covers the regulation of expression of CUP-assembled fimbriae in Gram-negative bacteria and uses selected examples to demonstrate both dedicated and global regulatory mechanisms

Bioengineering of non-pathogenic Escherichia coli to enrich for accumulation of environmental copper

Heavy metal sequestration from industrial wastes and agricultural soils is a long-standing challenge. This is more critical for copper since copper pollution is hazardous both for the environment and for human health. In this study, we applied an integrated approach of Darwin's theory of natural selection with bacterial genetic engineering to generate a biological system with an application for the accumulation of Cu2+ ions. A library of recombinant non-pathogenic Escherichia coli strains was engineered to express seven potential Cu2+ binding peptides encoded by a 'synthetic degenerate' DNA motif and fused to Maltose Binding Protein (MBP). Most of these peptide-MBP chimeras conferred tolerance to high concentrations of copper sulphate, and in certain cases in the order of 160-fold higher than the recognised EC50 toxic levels of copper in soils. UV-Vis spectroscopic analysis indicated a molar ratio of peptide-copper complexes, while a combination of bioinformatics-based structure modelling, Cu2+ ion docking, and MD simulations of peptide-MBP chimeras corroborated the extent of Cu2+ binding among the peptides. Further, in silico analysis predicted the peptides possessed binding affinity toward a broad range of divalent metal ions. Thus, we report on an efficient, cost-effective, and environment-friendly prototype biological system that is potentially capable of copper bioaccumulation, and which could easily be adapted for the removal of other hazardous heavy metals or the bio-mining of rare metals

Campylobacter jejuni bile exposure influences outer membrane vesicles protein content and bacterial interaction with epithelial cells

Abstract Campylobacter jejuni is a prevalent human pathogen and a major cause of bacterial gastroenteritis in the world. In humans, C. jejuni colonizes the intestinal tract and its tolerance to bile is crucial for bacteria to survive and establish infection. C. jejuni produces outer membrane vesicles (OMVs) which have been suggested to be involved in virulence. In this study, the proteome composition of C. jejuni OMVs in response to low concentration of bile was investigated. We showed that exposure of C. jejuni to low concentrations of bile, similar to the concentration in cecum, induced significant changes in the protein profile of OMVs released during growth without affecting the protein profile of the bacteria. This suggests that bile influences a selective packing of the OMVs after bacterial exposure to low bile. A low concentration of bile was found to increase bacterial adhesion to intestinal epithelial cells, likely by an enhanced hydrophobicity of the cell membrane following exposure to bile. The increased bacterial adhesiveness was not associated with increased invasion, instead bile exposure decreased C. jejuni invasion. OMVs released from bacteria upon exposure to low bile showed to increase both adhesion and invasion of non-bile-exposed bacteria into intestinal epithelial cells. These findings suggest that C. jejuni in environments with low concentrations of bile produce OMVs that facilitates colonization of the bacteria, and this could potentially contribute to virulence of C. jejuni in the gut