Research Training Proposal:
Gene expression of Vibrio vulnificus in mice

[Selected excerpts]


SPECIFIC AIMS

Vibrio vulnificus is a natural inhabitant of estuarine environments and is the leading cause of reported death from consumption of raw seafood, as well as a major cause of wound infection (1). Patients infected with V. vulnificus can die from sepsis within 24 hours of developing symptoms (1-3). One of the major steps of pathogenesis, replication in the host, is often overlooked when studying the virulence of this organism. Previously, the Gulig laboratory demonstrated that V. vulnificus proliferates extremely rapidly in host tissues, with a doubling time of 10 to 15 minutes in a mouse model (4). Very little is known about the cellular mechanisms that sustain such rapid growth. Thus, the goal of the proposed research is to begin to understand the fundamental metabolic pathways that allow V. vulnificus to elicit such a rapid and debilitating disease process.

It is likely that host tissues provide high-energy metabolites that are readily utilized by V. vulnificus, or that certain components of host tissue, when broken down by the bacterium, provide compounds that are normally synthesized de novo. Metabolic genes are often tightly regulated in such a way that they are only highly expressed when specifically needed. Therefore, we hypothesize that V. vulnificus modulates expression of genes involved in the metabolism of three key substrates found in fatty subcutaneous tissues – lipids, fatty acids and carbohydrates – to replicate extraordinarily quickly in the host. We will focus our efforts on genes encoding the most important bacterial proteins involved in this process: the global regulatory proteins responsible for modulating metabolic gene expression, the secreted degradative enzymes (e.g. lipases) responsible for converting host tissue into energy-rich substrates, and the high affinity transporters responsible for uptake of these substrates. Once critical metabolic pathways and genes are identified, we will examine their functions using a classical molecular pathogenesis approach.

The following two Specific Aims have been established to accomplish our goal:

Aim 1.
Use microarray technology to identify metabolic pathways induced during rapid in vivo growth of V. vulnificus in host tissues.
Aim 2.
Use a molecular pathogenesis approach to examine the functions of specific genes and their respective pathways identified in Aim 1 to identify the mechanisms responsible for the rapid disease process of V. vulnificus.


BACKGROUND AND SIGNIFICANCE

V. vulnificus causes two distinct disease processes: primary septicemia resulting from consumption of uncooked seafood and wound infection resulting from exposure to contaminated seawater or seafood products (5). This organism poses a substantial threat to immunocompromised individuals and patients with iron imbalance such as hemochromatosis and liver disease (6). Infection of susceptible individuals by either route typically results in fever, chills, hypotension, and severe tissue damage, leading to bullous skin lesions and hemorrhagic necrosis (7, 8). The infection progresses quickly, often resulting in death or requiring amputation within 24 hours after symptoms appear.

Mortality rates from V. vulnificus infection are high, exceeding 50% from septicemia and 25% from wound infection (1). These numbers may be higher for 2005, as 14 cases of wound-associated V. vulnificus infection were reported to the CDC within two weeks following landfall of Hurricane Katrina in August (9). Thus, although the numbers of hospitalized cases are low (30 to 50 per year in the United States)(1), V. vulnificus is a serious human pathogen capable of causing rapid, debilitating illness.

A. Pathogenesis of V. vulnificus

Among the seven steps of pathogenesis, those under particular scrutiny in the Gulig lab are growth, damage and evasion of host defenses. V. vulnificus is truly a flesh-eating bacterium, thriving extraordinarily well in fatty subcutaneous tissues during wound infection and sepsis. In fact, the Gulig laboratory has determined that V. vulnificus replicates more rapidly in mouse tissue than does Escherichia coli in nutrient-rich LB broth, suggesting that V. vulnificus is able to acquire and utilize tissue components, such as lipids, proteins, carbohydrates, and plasma components. Nothing is known about the specific nutrients utilized by V. vulnificus in vivo or about the metabolic pathways that allow enhanced growth in the host. V. vulnificus increases vascular permeability during infection and the Gulig laboratory has demonstrated that the organism proliferates around the vasculature, suggesting that plasma leaking into tissues may be an important source of nutrients.

Damage to the host is another critical aspect of V. vulnificus pathogenesis. This organism invades host tissues and causes severe damage to subcutaneous tissues in particular. Several extracellular proteins have been implicated in this process, including a hemolysin/cytolysin and a metalloprotease which, when injected as purified protein into mice, result in rapid dermonecrosis, vascular effects, and death (10, 11). However, mutations of the metalloprotease (vvpE) and cytolysin (vvhA) genes do not attenuate virulence in a mouse model (12, 13). The reason for this discrepancy is unclear, but may be related to problems with the animal model or due to compensation by other virulence factors. It is fair to say that the virulence factors responsible for the extensive damage in the mouse model and the human host have not been fully identified.

Finally, V. vulnificus is quite adept at evading the host immune response. Because the disease process is so rapid, the bacteria do not encounter adaptive humoral or cell-mediated immunity. However, the bacteria evade innate immunity by resisting complement and by subverting phagocytic defenses at every step. The polysaccharide capsule, a well-established virulence factor of V. vulnificus, helps the bacterium resist opsonic and lytic activities of complement and avoid phagocytosis. V. vulnificus may kill neutrophils in host tissues and possibly disable their motility (14, 15), because patients’ neutrophil counts are never elevated even though massive numbers of V. vulnificus are found in the infection. Acquisition of iron sequestered by the host, via siderophore biosynthesis and transport, is a well established virulence strategy of this organism (16).

In this proposal, we have chosen to focus our efforts on the ‘growth and metabolism’ element of pathogenesis by examining V. vulnificus gene expression in vivo. It is becoming increasingly apparent that metabolic pathways, virulence factors, and regulatory circuits are intricately entwined. For example, the enzyme adenylate cyclase is likely involved directly or indirectly in almost every aspect of V. vulnificus pathogenesis. Production of cAMP indirectly mediates catabolite repression, which may be important for growth on the diverse nutrient sources encountered in vivo. Mutation of adenylate cyclase of V. vulnificus decreases hemolysin, cytolysin, and metalloprotease activity, as well as motility (17). Thus, identification of the metabolic pathways and regulatory networks that allow this organism to replicate rapidly in host tissues is an important step towards dissecting the overall mechanism of V. vulnificus pathogenesis.

B. Use of microarray technology to investigate Vibrio spp. pathogenesis.

Study of bacterial virulence was initially limited to cataloguing putative virulence factors among clinical strains. This was followed by molecular genetics in which virulence factors could be definitively identified by combinations of cloning and mutagenesis. However, critical questions still exist about the host-pathogen interaction, particularly regarding the environmental signals sensed by the pathogen at different times and places in the host, as well as the mechanisms used by the pathogen to sense these environment cues and respond by replicating, resisting host defenses, and causing damage. We now have tools available to examine global gene expression of pathogens under a variety of conditions in vitro, as well as in the animal host...



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