Small GTPases and Bacterial Virulence Strategies

As discussed above and seen throughout this volume, small Rho GTPases are utilized extensively by bacterial pathogens to usurp normal cellular processes as part of virulence. This is presumably because of their central role in cellular functions and their many diverse downstream effects. These virulence factors generally fall into two categories: toxins that bind to cellular surfaces and can be internalized into host cells and effector proteins that are injected via type III and type IV secretion systems directly into host cells , where they can then target appropriate G proteins. In general, the toxins kill the host cells (thus their name), whereas effectors modulate cellular effects that benefit the pathogen, such as mediating bacterial uptake into host cells (invasion). Each of these processes is discussed in extensive detail throughout the remainder of this volume, and thus the details of each of the processes will not be reiterated here. Instead, a more general overview of the processes as they relate to bacterial virulence is presented in the context of bacterial virulence strategies.

Perhaps the most daunting obstacle a pathogen faces when encountering a potential host is the epithelial barrier. At some points on the body such as skin, this barrier is so impermeable the only way through it is via a break (cut) or at the base of hair follicle sites. However, at other sites in the body such as most mucosal surfaces, the barrier must be more permeable, as the body needs to shunt nutrients and cells such as phagocytes back and forth. The tight seal between epithelial cells (tight junctions or zona occludens) is supported by a band of actin running around the peripheral apical surface of each cell. Because it is made of actin, small Rho GTPases play a key role in maintaining its integrity (and thus cell polarity). Not surprisingly, bacterial pathogenshavedevelopedvariouswaystodisrupt thisbarriertogainaccessto deeper tissue or cause diarrhea. Thus Rho GTPases make attractive targets for pathogens that require the breakdown of epithelial barriers. Such breakdown may inhibit the normal immune sampling mechanisms by disrupting the normal cytology, disrupting chemotactic recruitment of macrophages and neutrophils, disrupting normal nutrient uptake, and altering luminal fluid production. It is thought that triggering diarrhea enhances the removal of normal flora that normally compete with an incoming pathogen for limited nutrient supplies. Additionally, enhanced fluid secretion increases the spread of a diarrheagenic pathogen in the environment, allowing it to colonize many new individuals.

Mucosal surfaces are usually the first host surface pathogens contact. The ability of pathogens to successfully adhere to cellular surfaces is akey virulence attribute [12]. Adherence is usually mediated by various bacterial adhesins such as fimbriae, pili, and afimbrial adhesins. Epithelial cells of the intestinal surface (and most other epithelial surfaces) form extensive networks of actin-based microvilli. Although this increases the cellular surface area, which enhances nutrient uptake, it is unclear whether this is a benefit or detriment to bacterial adherence. Nonetheless, some bacterial pathogens trigger microvil-lus disruption and surface rearrangements, including Salmonella species and the attaching and effacing pathogens such as pathogenic E. coli [6]. Because actin is the key regulator of microvilli, it is not surprising that these pathogens target small Rho GTPases [8] to disrupt microvilli. In addition, Salmonella is able to insert a phosphatidyl inositate phosphate phosphatase (SigD) into the host cell, which "loosens" the cellular surface (presumably by altering the underlying cytoskeletal architecture), enhancing bacterial invasion [17]. Given their key role in cellular signaling, the ability to modulate a host cellular surface could be of significant benefit to a pathogen intent on adhering to an epithelial surface. This could include up- or downregulating one or more host cell proteins with which the pathogen might interact (see chapter by Dumenil and Nassif, this volume). It could also include altering cellular surface function that may impact on the host's response to a pathogen (such as affecting Toll-like receptor signaling normally used to detect pathogens). Finally, it could also include altering the underlying cytoskeletal architecture. For example, Rho GTPases modulate the ezrin/radixin/moesin proteins in leukocytes, which affects integrin, ICAM, L-selectin, and other surface markers, which ultimately affects leukocyte migration, T-cell interaction with antigen-presenting cells, apoptosis, and phagocytosis [13].

Perhaps one of the most remarkable attributes of pathogens that activate small G proteins is that they often also encode the machinery to turn the G proteins off once the process is complete. This has been worked out very well for Salmonella species (see chapter by Schlumberger and Hardt, this volume, and [8]). This pathogen uses a type III secretion system to inject at least three bacterial effectors [SopE (a GEF), SopE2 (also a GEF), and SigD], which activate Cdc42 and Rac to facilitate actin-based bacterial uptake and invasion. However, it uses the same secretion system to also insert SptP, a GAP that inactivates Cdc42 and Rac activity and returns the actin cytoskeleton to a near-normal state after bacterial invasion, while the bacteria remain inside a vacuole in the host cell.

The ability to enter into a host cell provides a unique opportunity for a pathogen. It allows it to enter a noncompetitive environment free from normal flora. (In the large bowel, there is such competition for nutrients that E. coli's division time is about 24 h, compared to 20 min in rich broth, and it is in stationary phase in the gut). In addition, once inside a host cell, a pathogen is privy to a rich nutrient and moisture source, free from circulating antibodies and patrolling macrophages and neutrophils. Many bacterial pathogens invade host cells, and nearly all do this by exploiting members of the Rho GTPase family [5]. Most, such as Shigella and Salmonella, do this by injecting effectors that modulate Rac and Cdc42 activity. Examples of pathogens that use Rho family-mediated invasion include Bartonella, Brucella, Chlamydia, Listeria, and Pseudomonas. Indeed, it is felt that nearly all invasive pathogens use such a strategy for invasion, except perhaps for Rickettsia, which seems to use a phospholipase activity to break into a cell.

Once inside a cell, some pathogens digest the vacuole surrounding them and escape into the cytoplasm, where they then polymerize cytosolic actin [4]. This polymerization event propels them around inside the host cell and enables them to spread into adjacent host cells without becoming extracellular. Shigella, Listeria, and some Rickettsia use such a mechanism as part of their virulence [4]. However, instead of activating small Rho GTPases, these pathogens actually mimic downstream components to achieve this actin rearrangement. The Shigella protein IcsA recruits and activates N-WASP, leading to Arp2/3 complex recruitment and actin polymerization. This thus replaces the function of Cdc42. The Listeria protein ActA activates Arp2/3 directly, thereby directly mimicking N-WASP's activity.

Another family of pathogens, including enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), uses a type III system to insert effectors and their own receptors into epithelial surfaces. This results in a spectacular rearrangement of cellular actin and pedestal formation beneath the pathogen. Although Rho G proteins do not seem to be involved in this process [2], EPEC inserts its own protein (Tir) into host membranes, where it is tyrosine phosphorylated and binds the adaptor protein Nck, which then activates N-WASP and Arp2/3 complex recruitment [9]. The above examples indicate that modification or activation of Rho G proteins is not necessary, but instead mimics can be deployed to modulate actin dynamics.

For pathogens that invade and remain within a vacuole, it is critical to avoid lysosomal fusion (and subsequent bacterial death). Again, Rho GTPases (as well as Rabs) are involved in this process and other intracellular killing mechanisms, and inactivation of these may enhance intracellular survival (see chapter by Diebold and Bokoch, this volume). For example, Legionella pneumophila uses a type IV secretion system to insert an effector (RalF) into host cells that functions as a GEF to activate the small GTPase ARF, which modulates lysosomal fusion of the vacuoles containing bacteria [15].

Professional phagocytic cells are designed to use actin-mediated events to internalize pathogens and then destroy them in phagolysosomes. However, several pathogens including Yersinia, Pseudomonas, and EPEC have developed strategies to avoid phagocytosis by blocking this process [3]. Given the central role of actin in phagocytosis, it is no surprise that these mechanisms involve pathways to disrupt normal actin-mediated processes (see chapter by Deckert et al., this volume).

Many bacterial toxins and effectors either activate or deactivate Rho GTP-ases for a variety of functions [1]. Often this activity is mediated by enzymatic modification of the G protein. The variety of effects modulated by these bacterial toxins is impressive and serves as a major arsenal for pathogenic microbes. Often killing the host cell is one outcome, but many more subtle effects have been noted, which are a significant focus of this volume. In addition, these effectors and toxins have become valuable tools to dissect normal cellular functions and are used extensively by cell biologists without realizing their natural role in biology.

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