Research Interests

Our research interests center on the interaction of bacterial pathogens with their hosts. In particular, we are studying the molecular mechanisms by which the enteric pathogens Salmonella spp. and Campylobacter spp. interact with their host cells. Together, Salmonella spp. and Campylobacter spp. are responsible for the vast majority of food-borne illnesses in the United States and the rest of the world. Although most

Figure 1 (click to enlarge)

often these bacteria cause self-limiting gastroenteritis, they can also cause life-threatening disease such as typhoid fever. To study these bacterial pathogens, we take a multidisciplinary approach including bacterial genetics, cell biology, immunology, and structural biology.

Salmonella enterica

An essential feature of the pathogenicity of Salmonella is their ability to engage the host cell in a two-way biochemical interaction, or cross-talk. This cross-talk leads to responses from both the bacteria and the host-cell. Salmonella responds to the presence of the host-cell by activating a specialized protein secretion system termed Type III or contact dependent that is encoded within a pathogenicity island located at centisome 63 of the Salmonella chromosome (Figure 1). This protein secretion system is composed of a remarkable organelle, termed the needle complex (Figure 2). The architecture of the needle complex resembles that of the flagellar hook basal body complex, suggesting an evolutionary relationship between these two structures. The needle complex spans both the inner and outer membranes of the bacterial envelope. It is composed of two pairs of inner and outer rings that presumably anchor the structure to the inner and outer membranes of the bacterial envelope. The rings are connected by a rod-like structure which together form the base of the needle complex. A needle-like structure of ~80 nm in length protrudes outward from the base of the needle complex. The entire structure is ~100 nm in length and ~40 nm in diameter at its widest section.

Proteins destined to travel through the type III secretion pathway posses discrete signals that target them to the secretion apparatus.

Figure 3(click to enlarge)

In at least some type III secreted proteins, the secretion signal is located within the first ~15 amino acids or codons of the secreted polypeptide. In addition to the secretion mediated by the amino terminal sequence, another mechanism of secretion involves a family of customized chaperones. Although quite diverse at the primary amino acid sequence level, these chaperones share a number of biochemical properties such as small size (<20,000 kDa), low isoelectric point and a predominantly helical secondary structure. The recent determination of the crystal structure of the type III secreted protein of S. enterica SptP bound to its cognate chaperone SicP has greatly clarified the role of customized chaperones in the secretion process (Fig 3). The crystal structure showed that several copies of SicP bind to SptP maintaining it in an extended, non-globular conformation that yet retains its secondary structure. This finding suggests that the function of at least some type III secretion-associated chaperones may be to maintain their target proteins in a secretion competent state, which would be one of an unfolded polypeptide with secondary structural features that may be essential for their recognition by the secretion machinery.

The centisome 63 type III secretion system of Salmonella directs the export of several proteins. Some of these proteins transiently assemble in appendage-like structures called invasomes (Figure 4) while others are translocated into the host cell where they trigger or interfere with host-cell signal transduction pathways leading to a variety of responses. These responses are dependent on the type of infected cell. In non-phagocytic cells, Salmonella induces changes in the host-cell plasma membrane, and profound cytoskeletal rearrangements (Figure 5) that closely resemble the membrane ruffles induced by a variety of agonists, such as various hormones and growth factors, as well as the activation of cellular oncogenes. Membrane ruffling is accompanied by macropinocytosis (Figure 6), ultimately leading to bacterial internalization. A time-lapse video sequence of Salmonella-induced membrane ruffling can be seen in our movie page.

Another response induced by Salmonella in epithelial cells is the activation of various transcription factors which ultimately result in the production of pro-inflammatory cytokines such as IL-8. This is an important event in Salmonella pathogenesis, since one of the hallmarks of salmonellosis is the stimulation of a profuse inflammatory diarrhea. On the other hand, in macrophages Salmonella induces cytotoxic effects characterized initally by a rapid inhibition of membrane ruffling and macropinocytosis, followed by the induction of apoptotic cell death (Figure 7). A time-lapse sequence of Salmonella-induced macrophage cytotoxicity can be seen in our movie page. We are interested in understanding both the host-cell signal transduction pathways triggered by Salmonella, as well as the bacterial determinants that are responsible for triggering these signaling pathways.

We have found that at the center of the signaling pathways triggered by Salmonella that result in both nuclear and cytoskeletal responses, is the function of a group of small GTP-binding proteins of the Rho subfamily, in particular CDC42 and Rac. Expression of dominant negative forms of CDC42 (CDC42N17) and to a lesser extent Rac-1 (Rac-1N17) results in the complete abrogation of both Salmonella-induced cytoskeletal and nuclear responses. We have also found that the nuclear responses induced by Salmonella are the result of the activation of the transcription factors NF-kB and AP-1, as a consequence of the stimulation of the MAP kinase modules ERK, JNK and p38. A summary of the proposed Salmonella-induced signaling pathways is described in this diagram.

Figure 6(click to enlarge)

Work in our laboratory has led to a good understanding of the mechanisms by which Salmonella stimulates cellular responses. Upon contact with host cells, Salmonella injects, via its type III secretion system, a number of bacterial effector proteins. One of the bacterial proteins responsible for the stimulation of cellular responses, termed SopE, acts as an exchange factor for Rho GTPases, including CDC42 and Rac. Other effectors delivered by S. typhimurium into the host cell include an inositol phosphate phosphatase (SopB), and an actin binding protein (SipA). SopB stimulates signal transduction pathways that lead to Cdc42-dependent cellular responses. SipA modulates bacterial entry by spatially restricting the cytoskeletal rearrangements that result from the activation of Rho GTPases during S. typhimurium infection. It exerts its effect by decreasing the critical concentration of actin, stabilizing actin filaments and increasing the bundling activity of the actin binding protein plastin. This stabilizing function promotes a more pronounced outward extension of membrane ruffles and filopodia, thereby facilitating bacterial uptake.

The cellular responses induced by Salmonella are short lived. Shortly after bacterial infection, host cells regain their normal morphology. This phenomenon is mediated by SptP. Upon delivery into the host cell by the type III secretion system, this protein downmodulates the bacterial-induced cellular responses by acting as a GTPase-activating protein (GAP) for CDC42 and Rac. Thus, S. typhimurium modulates the actin cytoskeleton by alternatively activating and downmodulating the function of Rho GTPases, and by influencing different stages in the formation of membrane ruffles. This constitutes a remarkable example of pathogen evolution modulating host cellular functions. For a diagram summarizing the Salmonella invasion process, click here.

Further characterization of the mechanism of action as well as the determination of the crystal structures of these bacterial effector molecules have led us to advance the concept of structural and functional mimicry in bacterial virulence. In some cases, mimicry is achieved through virulence factors that are direct homologs of host proteins (e. g. the tyrosine phosphatase domain of SptP)(Fig. 7). In others, convergent evolution has produced new effectors that, while having no obvious amino-acid sequence similarity to host factors, are revealed by structural studies to display mimicry at the molecular level (e. g. the GAP domain of SptP)(Fig. 8).
A number of laboratories have made use of avirulent strains of S. typhimurium endowed with the ability to express cloned genes from other pathogens to stimulate immune responses against the recombinant protective antigens. Following oral inoculation, these organisms are known to invade and proliferate in the gut-associated lymphoid tissue (GALT). Therefore, most researchers have focused on the use of avirulent Salmonella vectors to stimulate mucosal immune responses to heterologous antigens. Efficient protection against infectious agents, however, often requires the action of both humoral and cellular immune mechanisms. An ideal antigen delivery system should therefore be capable of stimulating all branches of the immune response, in particular if such a delivery vector is to be

Figure 8(click to enlarge)

used to construct polyvalent vaccines. This is particularly so when the pathogen in question is a virus or an intracellular bacterium. In many of these cases, class I restricted immune responses are thought to be crucial for protection. S. typhimurium has the ability to invade mammalian cells. Unlike other facultative intracellular pathogens such as Listeria or Shigella spp., which gain access to the cytosol shortly after entry, Salmonella spp. remain inside the endocytic vesicle throughout their entire intracellular life cycle. Consequently, class I-restricted immune responses against heterologous antigens carried by Salmonella are generally poor. We have constructed avirulent strains of Salmonella with improved Class I antigen presenting capacity by using the invasion-associated Type III secretion apparatus to deliver epitopes into the host cell cytosol as a way of generating potent Class I restricted immune responses. We are using this approach to develop vaccines against many important pathogens. These studies may not only lead to the development of better antigen delivery systems but also to the understanding of the biological bases of persistent infection commonly observed with a variety of Salmonella serotypes.

Campylobacter jejuni

Campylobacter jejuni encodes a cytolethal distending toxin (CDT) that causes cells to arrest in the G2/M transition phase of the cell cycle (Fig. 9). Highly related toxins are also produced by other important bacterial

Figure 9(click to enlarge)

pathogens. CDT activity requires the function of three genes, cdtA, cdtB and cdtC. We have established that CdtB is the active subunit of CDT exerting its effect as a nuclease that damages the DNA and triggers cell cycle arrest. Microinjection of CdtB into target led to G2/M arrest and cytoplasmic distention, in a manner indistinguishable from CDT treatment. We also showed that when applied individually, purified CdtA, CdtB or CdtC do not exhibit toxic activity. In contrast, when combined, CdtA, CdtB and CdtC interact with one another to form an active tripartite holotoxin that exhibits full cellular toxicity. Therefore, CDT is an AB2-type toxin composed of CdtB as the enzymatically active (A) subunit, and CdtA and CdtC as the heterodimeric B subunit.