Induction and Evasion of Neutrophil Extracellular Traps by Campylobacter jejuni and its Implication in Disease

Campylobacteriosis, the foodborne disease caused by Campylobacter spp., infects one out of 10 individuals every year. C. jejuni accounts for 90% of these infections resulting in numerous postinfectious disorders including the development of colorectal cancer, Guillain-Barré syndrome, irritable bowel syndrome, and reactive arthritis. Despite its large impact on human health, the host immune response to the bacterium is largely uncharacterized. Chapter two of this dissertation addresses the development of neutrophil extracellular traps (NETs) within human and ferret campylobacteriosis. We observed NET-associated proteins increase in the feces of C. jejuni-infected patients and that in vitro C. jejuni induces NETs, which are cytotoxic to colonic epithelial cells. Furthermore, we observed NET-like structures within intestinal tissue of C. jejuni-infected ferrets where C. jejuni colonizes and causes tissue pathology. The work presented in chapter three of this dissertation characterizes a novel secreted C. jejuni effector, a sirtuin-like protein (SliP), which we found induces NETs. SliP is secreted into neutrophils, where it deacetylates proteins, specifically histone H3. As histone H3 acetylation plays a crucial role in chromatin structure and host transcription, activity of SliP could have immense impacts on the host cell physiology. Leveraging the murine model of campylobacteriosis, we determined a C. jejuni sliP mutant efficiently infects mice, but induction of proinflammatory cytokines and gastrointestinal pathology is decreased compared to wild-type C. jejuni infected mice demonstrating the importance of this protein in immune pathology in vivo. Finally, the work done in chapter four of this dissertation sought to characterize a C. jejuni nuclease (MugA) and its ability to assist in NET evasion. In this work, we performed a transposon screen of C. jejuni for nuclease production. We discovered that a mugA mutant was sensitive to NET killing and lacked nuclease activity. Interestingly, MugA degraded NETs lead to inflammasome activation within macrophages, and subsequent host inflammation. In conclusion, this dissertation demonstrates the influence of NETs within campylobacteriosis and highlights a potential target for therapeutic strategies. By specifically targeting SliP and MugA in C. jejuni for their role in immune modulation, these could be attractive therapeutics to reduce host inflammation and potential post-infectious disorder onset

The quorum-sensing regulation of Vibrio fischeri : novel components of the autoinducer/LuxR regulatory circuit

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution June 1999In the marine bacterium Vibrio fischeri two intercellular homoserine-Iactone signal molecules (luxI-dependent 30C6-HSL and the ainS-dependent C8-HSL) and the transcriptional activator LuxR regulate the luminescence system in a cell-density dependent manner by a process termed quorum sensing. In this study, five additional proteins whose production is regulated by quorum sensing are described, and the genes encoding four of the five proteins, denoted as QsrP, RibB, QsrV, and AcfA, are analyzed. Each protein is positively regulated by 30C6-HSL and LuxR and negatively regulated at low population density by C8-HSL. Probable LuxR/autoinducer binding sites are found in the promoter region of each. QsrP and RibB are encoded monocistronically, whereas AcfA and QsrV appear to be encoded by a two-gene operon. On the basis of sequence similarity to proteins of known function from other organisms, RibB is believed to be an enzyme that catalyzes the transformation of ribulose 5-phosphate to 3,4-dihydroxy-2- butanone 4-phosphate, a precursor for the xylene ring of riboflavin; AcfA is believed to be a pilus subunit; and the functions of QsrP and QsrV are unknown at this time. A qsrP mutant was reduced in its ability to colonize its symbiotic partner, Euprymna scolopes when placed in competition with the parent strain. On the other hand, a mutant strain of V. fischeri containing an insertion in acfA, which is believed to be polar with respect to qsrV, displayed enhanced colonization competence in a competition assay. A ribB mutant grew well on media not supplemented with additional riboflavin and displayed normal induction of luminescence. Both phenotypes suggest that the lack of a functional ribB gene is complemented by another gene of similar function in the mutant. Oriented divergently from acfA are open reading frames that code for two putative proteins that are similar in sequence to members of the LysR family of transcriptional regulators. Organization of the two divergent sets of genes and the shared promoter region suggests that transcription of acfA and qsrV may be regulated by one or both of these divergently transcribed proteins. This work defines a quorum-sensing regulon in V. fischeri. A model describing its regulation is presented.Woods Hole Oceanographic Institution, including The J. Seward Johnson Fund, for contributing financiall

Maintenance of heterocyst patterning in a filamentous cyanobacterium

In the absence of sufficient combined nitrogen, some filamentous cyanobacteria differentiate nitrogenfixing heterocysts at approximately every 10th cell position. As cells between heterocysts grow and divide, this initial pattern is maintained by the differentiation of a single cell approximately midway between existing heterocysts. This paper introduces a mathematical model for the maintenance of the periodic pattern of heterocysts differentiated by Anabaena sp. strain PCC 7120 based on the current experimental knowledge of the system. The model equations describe a non-diffusing activator (HetR) and two inhibitors (PatS and HetN) that undergo diffusion in a growing one-dimensional domain. The inhibitors in this model have distinct diffusion rates and temporal expression patterns. These unique aspects of the model reflect recent experimental findings regarding the molecular interactions that regulate patterning in Anabaena. Output from the model is in good agreement with both the temporal and spatial characteristics of the pattern maintenance process observed experimentally

The quorum-sensing regulation of Vibrio fischeri : novel components of the autoinduce/LuxR regulatory circuit

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1999.Includes bibliographical references.by Sean M. Callahan.Ph.D

Draft Genome Sequence of Vibrio coralliilyticus strain OCN008 Isolated from Kāneʻohe Bay, Hawaiʻi.

Vibrio coralliilyticus is a Gram-negative bacterium found in seawater and is associated with diseased marine organisms. Strains of V. coralliilyticus have been shown to infect coral from multiple genera. We report the draft genome sequence of V. coralliilyticus strain OCN008, the third V. coralliilyticus genome to be sequenced

ABC Transporter Required for Intercellular Transfer of Developmental Signals in a Heterocystous Cyanobacterium

In the filamentous cyanobacterium Anabaena, patS and hetN encode peptide-derived signals with many of the properties of morphogens. These signals regulate the formation of a periodic pattern of heterocysts by lateral inhibition of differentiation. Here we show that intercellular transfer of the patS- and hetN-dependent developmental signals from heterocysts to vegetative cells requires HetC, a predicted ATP-binding cassette transporter (ABC transporter). Relative to the wild type, in a hetC mutant differentiation resulted in a reduced number of heterocysts that were incapable of nitrogen fixation, but deletion of patS or hetN restored heterocyst number and function in a hetC background. These epistasis results suggest that HetC is necessary for conferring self-immunity to the inhibitors on differentiating cells. Nine hours after induction of differentiation, HetC was required for neither induction of transcription of patS nor intercellular transfer of the patS-encoded signal to neighboring cells. Conversely, in strains lacking HetC, the patS- and hetN-encoded signals were not transferred from heterocyst cells to adjacent vegetative cells. The results support a model in which the patS-dependent signal is initially transferred between vegetative cells in a HetC-independent fashion, but some time before morphological differentiation of heterocysts is complete, transfer of both signals transitions to a HetC-dependent process

Assessment of disease lesion removal as a method to control chronic Montipora white syndrome

Coral colonies in Ka–ne‘ohe Bay, Hawai‘i (USA), are afflicted with the tissue loss disease chronic Montipora white syndrome (cMWS). Here we show that removal of chronic disease lesions is a potential method to slow the progression of cMWS in M. capitata. Over the 24 wk observation period, treatment colonies lost almost half the amount of tissue that was lost by control colonies. The percentage of tissue loss at each sampling interval (mean ± SEM; treatment: 1.17 ± 0.47%, control: 2.25 ± 0.63%) and the rate of tissue loss per day (treatment: 0.13 ± 0.04%, control: 0.27 ± 0.08%) were both significantly lower on treated colonies than control colonies. While lesion removal stopped tissue loss at the initial infection site, which allowed colony healing, it did not prevent re-infection; in all but one of the treated colonies, new cMWS lesions appeared in other areas of the colony but not around the treatment margins. Additionally, the rate of new infections was similar between treatment and control colonies, indicating that physical injury from lesion removal did not appear to increase cMWS susceptibility. These results indicate that lesion removal reduced morbidity in M. capitata exhibiting cMWS but did not stop the disease

Complete Genome Sequence of Vibrio coralliilyticus Strain OCN014, Isolated from a Diseased Coral at Palmyra Atoll

Vibrio coralliilyticus is a marine gammaproteobacterium that has been implicated as an etiological agent of disease for multiple coral genera on reefs worldwide. We report the complete genome of V. coralliilyticus strain OCN014, isolated from a diseased Acropora cytherea colony off the western reef terrace of Palmyra Atoll

Complete Genome Sequence of Pseudoalteromonas sp. Strain OCN003, Isolated from Kāneʻohe Bay, Oʻahu, Hawaii

Pseudoalteromonas sp. strain OCN003 is a marine gammaproteobacterium that was isolated from a diseased colony of the common Hawaiian reef coral, Montipora capitata, found on a reef surrounding Moku o Loʻe in Kāneʻohe Bay, Hawaii. Here, we report the complete genome of Pseudoalteromonas sp. strain OCN003

First Record of Black Band Disease in the Hawaiian Archipelago: Response, Outbreak Status, Virulence, and a Method of Treatment

A high number of coral colonies, Montipora spp., with progressive tissue loss were reported from the north shore of Kaua‘i by a member of the Eyes of the Reef volunteer reporting network. The disease has a distinct lesion (semi-circular pattern of tissue loss with an adjacent dark band) that was first observed in Hanalei Bay, Kaua‘i in 2004. The disease, initially termed Montipora banded tissue loss, appeared grossly similar to black band disease (BBD), which affects corals worldwide. Following the initial report, a rapid response was initiated as outlined in Hawai‘i’s rapid response contingency plan to determine outbreak status and investigate the disease. Our study identified the three dominant bacterial constituents indicative of BBD (filamentous cyanobacteria, sulfate-reducing bacteria, sulfide-oxidizing bacteria) in coral disease lesions from Kaua‘i, which provided the first evidence of BBD in the Hawaiian archipelago. A rapid survey at the alleged outbreak site found disease to affect 6-7% of the montiporids, which is higher than a prior prevalence of less than 1% measured on Kaua‘i in 2004, indicative of an epizootic. Tagged colonies with BBD had an average rate of tissue loss of 5.7 cm2/day over a two-month period. Treatment of diseased colonies with a double band of marine epoxy, mixed with chlorine powder, effectively reduced colony mortality. Within two months, treated colonies lost an average of 30% less tissue compared to untreated controls