Bacterial-Enterocyte Crosstalk: Cellular Mechanisms in Health and Disease

INTRODUCTION The human colon maintains a microbial density approaching 1012 organisms per gram of feces, representing a perfectly balanced ecosystem. The commensal microflora, derived from the Latin “commensalis” meaning “at table together”, consists of more than 400 species and lives in perfect harmony with the human intestine (1). Recently the term probiotics was defined by Fuller (2) as a “live microbial food supplement which beneficially affects the host animal by improving its intestinal microbial balance”. Commercial probiotics have been used extensively in the prevention and treatment of human conditions from diarrhea of the infant to inflammatory bowel disease (3). However, the mechanisms by which these intestinal bacteria prevent an inflammatory response in reaction to the plethora of foreign antigens available in the intestinal lumen have just begun to come under investigation. In contrast to the myriad of indigenous microorganisms in symbiosis it takes just 10 to 100 single organisms of the pathogen Shigella to destroy this peaceful coexistence and result in bloody mucoid diarrhea associated with fever, nausea, and abdominal cramps. This infectious disease still costs more than half a million people their lives every year and most of them are children under 5 years of age. Even in a developed country such as the U.S.A., bacterial food-borne pathogens may cause an estimated 4.1 million illnesses, 45,000 hospitalizations, and 1,500 deaths every year (4). This continuing health threat has resulted in the development of the Foodborne Diseases Active Surveillance Network (Foodnet) by the Centers for Disease Control and Prevention (CDC). To maintain health and cause disease, which are both active processes, bacteria and the intestinal epithelium must actively interact. This interaction is referred to as “crosstalk” and this research area has become one of the most active in gastroenterology today. Commensal microflora together with enterocytes are hypothesized to act as a gatekeeper to protect the human organism from penetration and disease. On the other hand, bacterial pathogens themselves have developed in their two and a half billion years on earth (5) sophisticated mechanisms, like the type III secretion system, to interact with host cells to promote their own survival. An understanding of the molecular mechanisms by which these microorganisms interact with the intestinal epithelium to promote health or cause disease is expanding rapidly. It is becoming more and more obvious that prokaryotic organisms manipulate the human enterocyte functions in many ways for their own benefit. In contrast commensal microflora and probiotics can provide benefits to the host beyond a local effect in the intestine. These organisms use similar mechanisms to that of the pathogen but these mechanisms are largely undefined because of a lack of sufficient basic research. The interaction of the bacterium with the intestinal epithelial cell (IEC) has, somewhat artificially, been divided into the following categories: i) attachment and invasion, ii) altered epithelial barrier function, and iii) inflammation. All these phenomena are constantly both activated and suppressed at the same time in the human gut by different microorganisms in a highly sophisticated interaction with IEC. In this review we will not comprehensively cover these topics but will rather highlight some of the more recent findings to illustrate the cellular mechanisms that effect microbial-enterocyte crosstalk in health and disease in the human intestine. ATTACHMENT AND INVASION Infectious diarrhea is still the leading cause of death in children in developing countries with the problem being most severe during the first year of life. It accounts for a total mortality of 3.3 million deaths per year (6). The enteropathogens Escherichia coli (E. coli) and Salmonella enterica are closely related and most experts consider Shigella to belong to the species of the genus of Escherichia. The considerable pathogenic diversity superimposed on a relatively conserved genetic backbone is a result of the evolution and dispersion of virulence genes that are spread between the species on transmissible genetic elements. These elements include plasmids, bacteriophages (viruses that infect bacteria), and pathogenicity islands (Table 1). Pathogenicity islands are defined as discrete and relatively large loci containing sets of virulence genes that are present within the genome of pathogenic bacteria but are absent from the genome in closely related non-pathogenic strains (7). Many pathogenicity islands encode specialized systems for the delivery of virulence proteins into the host cell. One such system is termed the type III secretion system (TTSS). TTSS are specialized virulence devices that have evolved to modify host cell function through the direct translocation of bacterial proteins into the host cytoplasm (8). The structure has a needle-like appearance in Shigella (9) and Salmonella (10) but is somewhat thicker as the appendage of enteropathogenic Escherichia coli (EPEC) (11). In all cases it bridges between bacterial and host cells.TABLE 1: Selected bacterial factors and their role in the bacterial interaction with the intestinal epitheliumSalmonella The enteric pathogen Salmonella causes a range of diseases from gastroenteritis to enteric (typhoid) fever. Typhoid fever is a protracted systemic illness that results from the exclusively human pathogens S. typhii and S. paratyphi. Without treatment mortality is between 10% and 15%. In contrast, the many nontyphoidal Salmonella strains, such as S. typhimurium and enteritidis, infect a wide range of animal hosts including poultry, cattle, and pigs and usually cause a self-limiting enteritis in humans. Very young, old, or immunocompromised individuals are prone to a systemic disease which causes 500 deaths in the U.S.A. alone every year (4). After entering the GI tract, via contaminated food or water, in a manner similar to other pathogens discussed in this review, Salmonella must penetrate the intestinal mucous layer in the small bowel before encountering and adhering to IEC. Salmonella expresses several fimbriae, which contribute to the adherence process (12). Salmonella typhimurium finally invades its animal host by entering and traversing the epithelial monolayer lining the intestine. This is achieved by initiating an extensive enterocyte-cytoskeletal rearrangement that results in membrane ruffling and bacterial internalization by epithelial cells, which are normally nonphagocytotic (13). Bacterial adhesion to the tips of apical microvilli activates the contact-dependent TTSS through which bacterial products are injected into the host cell (14). Recent research efforts have focused on identifying the responsible virulence proteins and describing host-cell function targeted by these proteins. Members of the Rho family, cell proteins involved in signal transduction, of small Guanine Triphosphatases (GTPases) which includes RhoA, Rac1, and Cdc42, play a central role in regulating the actin cytoskeleton (15). In in vitro studies it was first demonstrated that invasion of Salmonella typhimurium was primarily dependent on Cdc42. Expression of a point mutation on Cdc42 hinders it from binding GTP, which acts in an inhibitory manner preventing bacterial entry. Expression of dominant-negative Rac1 only partially inhibits internalization. In addition, Cdc42 mediates the Salmonella-induced activation of the signaling protein Jun kinase (JNK) (16). However, these studies were conducted in nonpolarized cells, which means that they do not have an apical side and basolateral side. This polarization seems to be a critical cellular characteristic for the intestinal epithelium since the composition of the cellular environment on both sides of the epithelial surfaces, the lumen and subepithelial space, are completely different. In a polarized model epithelium, which probably simulates the in vivo situation more accurately, only Rac1, but not Cdc42, significantly inhibited bacterial entry from the apical side of the host cell (17). It was determined that the modulation of Rho GTPase activity was dependent on two Salmonella effector proteins SopE and Spt (18,19,20). SopE was characterized as a guanine nucleotide exchange factor (GEF) by its ability to stimulate in vitro a nucleotide exchange on Cdc42, Rac1, and RhoA and is required for bacterial entry. A recently described similar bacterial protein, SopE2, possesses comparable activity (21,22). Other bacterial effector proteins, SipA and SipC, are also involved in regulating and timing the invasion process but do not seem to be essential (23,24). Shigella Shigella spp. is a group of gram-negative enteric bacilli that causes acute bacillary dysentery in humans. The syndrome caused by Shigella consists of painful abdominal cramps, nausea and fever, along with blood and mucus in the stool. In its most severe forms, shigellosis is associated with an intense inflammatory reaction that leads to the destruction of the colonic mucosa (25). Shigellosis is mostly a pediatric disease, with greater than 60% of the cases occurring in children of 1 to 5 years of age, and it is primarily a third-world disease, with approximately 150 million cases every year. Most important is a deadly disease causing about 1 million deaths every year, again primarily in infants and young children (26). The ability of this food-borne pathogen to invade and colonize the colonic epithelium is a key determinant in the establishment of the disease. The invasion capability of Shigella flexneri has been localized to a 31 kilobase (kb) region on its pathogenicity islands (27). This 31kb region codes for many closely linked genes, including the invasion plasmid antigen (Ipa), the membrane expression of invasion plasmid antigens (mxi), and the surface expression of invasion plasmid antigen (spa) genes as well as other, independently expressed genes. One astonishing feature about Shigella infection is that although very few bacteria (10 to 100) can cause dysentery in humans, this microorganism is completely nonpathogenic for experimental animals except certain primates. An additional characteristic of Shigella is that they are unable to invade epithelial cells from the apical surface and therefore most first gain access to the basolateral side before entry (28) (Fig. 1). 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