Question

Design the perfect bacterial pathogen. Include in your description why/how it is successful at transmitting from host to host. What types of tactics does it use to evade killing by the immune system? The perfect pathogen should have one novel virulence factor that you designed/created. Describe how this virulence factor fits in with the other strategies used by the bacteria, and how it facilitates the bacteria’s survival and spread.

Answer 1

Mycobacterium tuberculosis :

M. TUBERCULOSIS VIRULENCE AND THE DISEASE PROCESS

The previous section described the different stages of human TB at the level of the infected patient and the involved organ systems. This section summarizes events in infection from the cellular and molecular viewpoint of both the infecting bacterium and its host. An excellent review of host innate immunity and responses to M. tuberculosis infection has recently appeared in this journal (297), and to avoid repetition, some areas are mentioned only briefly.

Events in the Infectious Process

Early events.As discussed above, M. tuberculosis usually enters the alveolar passages of exposed humans in an aerosol droplet, where its first contact is thought be with resident macrophages, but it is also possible that bacteria can be initially ingested by alveolar epithelial type II pneumocytes. This cell type is found in greater numbers than macrophages in alveoli, and M. tuberculosis can infect and grow in these pneumocytes ex vivo (24, 190). In addition, dendritic cells play a very important role in the early stages of infection since they are much better antigen presenters than are macrophages (286) and presumably play a key role in activating T cells with specific M. tuberculosis antigens (31, 114). Since dendritic cells are migratory, unlike differentiated macrophages (164), they also may play an important role in dissemination of M. tuberculosis. However, this discussion is limited to the much more extensively studied and better understood M. tuberculosis-macrophage interaction. The bacteria are phagocytosed in a process that is initiated by bacterial contact with macrophage mannose and/or complement receptors (254). Surfactant protein A, a glycoprotein found on alveolar surfaces, can enhance the binding and uptake of M. tuberculosisby upregulating mannose receptor activity (107). On the other hand, surfactant protein D, similarly located in alveolae, inhibits phagocytosis of M. tuberculosis by blocking mannosyl oligosaccharide residues on the bacterial cell surface (90), and it is proposed that this prevents M. tuberculosisinteraction with mannose receptors on the macrophage cell surface. Cholesterol in cell plasma membranes is thought to be important for this process, since removal of this steroid from human neutrophils decreases the phagocytosis of M. kansasii (221) and similar depletion experiments prevented the entry of M. bovis BCG into mouse macrophages (106). The human toll-like receptor 2 (TLR2) also plays a role in M. tuberculosis uptake (201), and this important interaction with bacterial components is discussed later in this review. On entry into a host macrophage, M. tuberculosis and other intracellar pathogens initially reside in an endocytic vacuoule called the phagosome. If the normal phagosomal maturation cycle occurs, i.e., phagosome-lysosome fusion, these bacteria can encounter a hostile environment that includes acid pH, reactive oxygen intermediates (ROI), lysosomal enzymes, and toxic peptides. Reactive nitrogen intermediates (RNIs) produced by activated mouse macrophages are major elements in antimicrobial activity (197), and mice with mutations in the gene encoding the macrophage-localized cytokine-inducible nitric oxide synthase gene are more susceptible to various pathogens, including Leishmania major (311), Listeria monocytogenes(169), and M. tuberculosis (168). The M. tuberculosis result is consistent with the results of other experiments showing that RNIs are the most significant weapon against virulent mycobacteria in mouse macrophages (48, 50) and the observation that resistance to RNIs among various strains of M. tuberculosis correlates with virulence (48, 50, 202). The presence of RNIs in human macrophages and their potential role in disease has been the subject of controversy, but the alveolar macrophages of a majority of TB-infected patients exhibit iNOS activity (200).

Since most macrophage killing of bacteria occurs in the phagolysosome (89), intracellular pathogens have evolved many ways to avoid this hostile vacuolar microenvironment. Listeria and Shigellaphysically escape the phagosome and replicate in the cytoplasm (252), and Legionella inhibits phagosome-lysosome fusion (134). Salmonella enterica serovar Typhimurium phagosomes also are diverted from the normal endocytic pathway of phagosome-lysosoma fusion (42, 233), and this bacterium requires acidification of the phagosome to survive in macrophages (234). Pathogenic mycobacteria also inhibit phagosome-lysosome fusion (6, 98), but unlike the situation for Salmonella, the mycobacterial phagosome is not acidified (60). This is presumably due to the exclusion of proton ATPases from the mycobacterial phagosome (281), but it is not clear that the blocking of endosomal maturation is essential for M. tuberculosis survival in macrophages. Live M. tuberculosis cells can made to traffic to late endosomes by opsonization with polyclonal antibodies against M. tuberculosisH37Rv, which presumably directs bacterial binding to Fc receptors. However, this rerouting has no effect on bacterial growth in mouse peritoneal macrophages (6). On the other hand, a recent study in which human monocyte-derived macrophages (MDMs) were infected with M. tuberculosis Erdmann opsonized with a polyclonal antibody raised against the M. tuberculosis cell surface glycolipid lipoarabinomannan (LAM) showed that this treatment causes 80% loss of bacteria as well as increased trafficking to late, more acidic endosomes (175). The different results in these two experiments have not been resolved but could be in part due to the source of the macrophages, the nature of the antibodies, and the bacterial strains used. An interesting finding in the latter work is that Ca2+ signaling is inhibited when M. tuberculosis enters human macrophages but not when killed M. tuberculosis or antibody-opsonized M. tuberculosis cells are phagocytosed (175). This effect was correlated with trafficking to late endosomes; i.e., elevated Ca2+ levels were associated with phagolysosome formation. Since Ca2+ can stimulate many host responses to infection, e.g., the respiratory burst as well as NO and cytokine production, preventing increases in Ca2+ levels would help M. tuberculosis avoid these host defense mechanisms. It has also been postulated that a selective advantage to M. tuberculosis of staying in an early endosome is that there would be less host immunosurveillance by CD4+ T cells. In agreement with this idea, there is a decrease in the expression of major histocompatibility complex class II (MHC-I) proteins and in the MHC-II presentation of bacterial antigens in macrophages after M. tuberculosisinfection (201). As discussed below, this effect seems to be induced by presence of the secreted or surface-exposed M. tuberculosis 19-kDa lipoprotein, which is thought to interact with TLR2 in the early phase of bacterial entry into macrophages (287). The mechanism by which virulent mycobacteria prevent phagosomal maturation is not known, but in the normal maturation of the mycobacterial phagosome there is a successive recruitment of Rab proteins, which are small GTPases involved in endosome trafficking; i.e., Rab5 associates with early endosomes, and Rab7 is found in later endosomes. The M. tuberculosis phagosome that does contain Rab5 does not recruit Rab7 (298). Also, TACO, a member of the coronin family of actin binding proteins, is preferentially recruited to the mycobacterial phagosome of infected murine macrophages, where it was reported to be retained in phagosomes containing live and not killed M. bovis BCG (91). However, a more recent study, in which phagosomes and other macrophage organelles were isolated, has shown that the association of coronin with phagosomes containing live M. bovis BCG in both murine and human macrophages is transient and is retained only on phagosomes containing clumped bacteria (257). These latter results suggest that coronin is not involved with the arrest in phagosome trafficking observed in M. tuberculosis infections of macrophages. It is also not known whether the exclusion of Rab7 and/or the decreased Ca2+signaling discussed above is directly responsible for this block in phagosome maturation or is a secondary consequence.

Later events.The relative ease of working with tissue culture has provided many data on M. tuberculosis entrance and trafficking in the macrophage and on other responses of the infected cells, as discussed above, but there is much less information on how the bacterium survives and grows during later stages of infection in the lung. It is known that infected macrophages in the lung, through their production of chemokines, attract inactivated monocytes, lymphocytes, and neutrophils (297), none of which kill the bacteria very efficiently (89). Then, granulomatous focal lesions composed of macrophage-derived giant cells and lymphocytes begin to form. This process is generally an effective means of containing the spread of the bacteria. As cellular immunity develops, macrophages loaded with bacilli are killed, and this results in the formation of the caseous center of the granuloma, surrounded by a cellular zone of fibroblasts, lymphocytes, and blood-derived monocytes (63). Although M. tuberculosis bacilli are postulated to be unable to multiply within this caseous tissue due to its acidic pH, the low availability of oxygen, and the presence of toxic fatty acids, some organisms may remain dormant but alive for decades. The strength of the host cellular immune response determines whether an infection is arrested here or progresses to the next stages. This enclosed infection is referred to as latent or persistent TB and can persist throughout a person's life in an asymptomatic and nontransmissible state. In persons with efficient cell-mediated immunity, the infection may be arrested permanently at this point. The granulomas subsequently heal, leaving small fibrous and calcified lesions. However, if an infected person cannot control the initial infection in the lung or if a latently infected person's immune system becomes weakened by immunosuppressive drugs, HIV infection, malnutrition, aging, or other factors, the granuloma center can become liquefied by an unknown process and then serves as a rich medium in which the now revived bacteria can replicate in an uncontrolled manner. At this point, viable M. tuberculosis can escape from the granuloma and spread within the lungs (active pulmonary TB) and even to other tissues via the lymphatic system and the blood (miliary or extrapulmonary TB). When this happens, the person becomes infectious and requires antibiotic therapy to survive (63).

Currently, there is little information concerning how M. tuberculosis responds to the environment of the lung, preventing the development of rational strategies for treating latent and chronic infections as well acute manifestations of the disease. Some experiments on M. tuberculosis “persistence” or “latency” have been performed, using two chronic-infection models in mice in which bacteria can be maintained in a steady state in the absence of disease or are actually not cultivatible (reviewed in reference 95). However, it is still not certain whether bacteria in the chronic-disease models are actually viable but nongrowing, which would reflect a true latent state, or whether they are growing and dying at the same rate. The fact that M. tuberculosis in a chronically infected mouse model is susceptible to isoniazid (INH) (238), a drug that is effective only against growing M. tuberculosis(308), provides evidence for the latter explanation, i.e., balanced growth and death. There is biochemical evidence that the intermediary metabolism of M. tuberculosis changes, during the course of chronic mouse infections, from an aerobic, carbohydrate-metabolizing mode to one that is more anaerobic and lipid utilizing (258, 259). It is only in recent years that the full significance of these apparent changes in intermediary metabolism for acute or chronic infection has become apparent, as discussed later in this review.

Immune system evasion

Unlike other pathogens, MTB infects and resides within immune cells, this bacterium has the ability to live within the dynamic and heterogeneous environment of macrophage phagosome. Here, the bacilli use a plethora of strategies to evade the microbicidal mechanisms of macrophage, including: phagosome-lysosome fusion, recruitment of hydrolytic lysosomal enzymes, production of reactive oxygen/nitrogen species, antigen presentation and apoptosis. Disruption of those functions in turn disrupts the adaptive immune response. Phagocytosis is an active process that depends on the interaction with various surface receptors expressed on the macrophage such as complement receptor type 3 (CR3), FCγ receptors and lectin receptors and it can be opsonic or non-opsonic. However, non-opsonic phagocytosis of MTB results in higher intracellular survival, although it is difficult to assess if the engagement of specific receptor determines the course of infection TB uses PDIM lipids to evade detection by TLRs, thereby preventing mycobacterial delivery into microbicide macrophages expressing iNOS . Moreover, MTB actively blocks the phagosome maturation by their cell wall components or through the secretion of various macromolecules that interferes with this process, which enables bacterial survival in a non-acidified intracellular compartment