A plague upon the phagocytes

Plague bacteria are renowned for causing some of the most devastating epidemics in human history. We are now closer to understanding why: the pathogen selectively disarms key cells of the innate immune system, weakening the front-line defenses of the body.

Plague is arguably the leading cause of pathogen-related death in human history¹. Epidemics of bubonic plague—the Black Death—killed an estimated 200 million Europeans during the Middle Ages. Even today, there are isolated outbreaks, and a recent epidemic in Madagascar suggests that plague could re-emerge as a global threat to human health^1,2. To cause human infections, the pathogen Yersinia pestis must circumvent destruction by cells of the innate immune system, including phagocytes such as neutrophils and macrophages.

Understanding how this bacterium evades the front-line defenses of the immune system could shed light on how the onset of fatal disease occurs so rapidly that an acquired immune response fails to develop. Such understanding could also inform the development of a safe and effective vaccine for plague.

Recent studies in Science³ and Infection and Immunity⁴ bring us a step closer to resolving these important problems. In Science, Marketon et al. show that plague bacteria destroy specific cells of the innate immune system in vivo, and thereby survive and cause disease³. In a report from the same laboratory, Overheim et al. reveal a new plague vaccine strategy that protects from lethal challenge with the pathogen while reducing undesirable immunosuppressive side effects⁴.

Y. pestis is distributed throughout the world in rodent reservoirs, and is maintained by rodent-flea-rodent transmission cycles^1,2. Transmission from rodents to humans occurs typically by fleabite. Bacteria disseminate from bite to lymph nodes, producing severely inflamed and swollen lymph nodes called buboes—hence the name bubonic plague. Bubonic plague can be resolved with antibiotics, but will often result in fatal septicemia if left untreated. Spread of bacteria from blood to the lungs can lead to pneumonic plague, which is directly transmissible from person to person by aerosolized droplets².

A key component to developing better treatments for late-stage disease and a vaccine against plague is to gain an enhanced understanding of how the plague bacillus causes human disease. Because phagocytic cells are essential for defense against bacterial infections, the ability of plague bacilli to alter normal phagocyte function is almost certainly crucial to its success as a pathogen. Virulence of Y. pestis—like many other pathogens—depends on a needle-like complex known as a type III secretion system, which is used by the bacterium to deliver proteins into the cytoplasm of host cells⁵. Previous studies with other Yersinia species have shown that toxic proteins known as Yops (Yersinia outer proteins) trigger apoptosis and alter immune cell functions in vitro^1,2,5.

As a step toward understanding how Y. pestis causes plague, Marketon et al. developed an in vivo reporter system consisting of Yop fusion proteins transformed into Y. pestis³. The group evaluated targeting of Yops to immune cells in the spleen using a mouse model of septicemic plague. To their surprise, plague bacteria injected Yops into neutrophils, macrophages and dendritic cells, but not into B cells or T cells³. The end result was the pathogen avoided destruction and killed host cells that would normally prevent infection (Fig. 1). This observation is consistent with the rapid course of the disease—Y. pestis must quickly eliminate cells of the innate immune system to disseminate. Although previous studies have shown that bacteria related to the plague species can alter phagocyte function in vitro, the studies in Science reveal selective targeting and elimination of these crucial cells by plague-causing bacteria during mammalian infection.

Figure 1: Plague bacteria selectively target key cells of the innate immune system during infection by injecting Yops (Yersinia outer proteins).

Katie Ris

Inset, scanning electron micrograph of Y. pestis. SEM by Elizabeth Fischer, Rocky Mountain Microscopy Branch, Rocky Mountain Laboratories.

Notably, the types of immune cells to which Yops were delivered during animal infection in vivo differed markedly from those in vitro. In addition to phagocytes, B cells and CD4⁺ and CD8⁺ T cells were injected with Yops in vitro³, and the most frequently injected cells were B cells and dendritic cells³. These observations highlight the importance of using appropriate in vivo animal model systems to evaluate pathogenesis mechanisms.

In addition to their direct toxicity to innate immune cells, certain Yops act to suppress the inflammatory response⁵. In particular, the low-calcium response V antigen (LcrV), which has been tested successfully as a potential plague vaccine antigen in numerous animal model systems^6,7,8,9,10, elicits production of the anti-inflammatory cytokine interleukin (IL)-10 and reduces production of the proinflammatory cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α¹¹. These immunomodulatory properties present a potentially serious obstacle to the use of an LcrV-based vaccine.

To overcome that problem, Overheim et al. evaluated the vaccine efficacy of LcrV proteins containing sequential 30–amino acid deletions⁴. First, the group investigated the ability of the full-length and truncated LcrV proteins to trigger IL-10 release and thereby block proinflammatory capacity. After identifying truncated proteins that failed to inhibit proinflammatory cytokine release, they went on to test the ability of each to elicit a protective immune response against lethal challenge in mice⁴. One LcrV protein with a deletion near the C terminus (residues 271–300) protected animals from lethal plague infection and did not block proinflammatory capacity.

Despite these important discoveries, there are still significant gaps in our understanding of Y. pestis as a human pathogen. First, it is not known how plague bacteria selectively target phagocytes in vivo. Bliska et al. and Young et al. showed that Yersinia proteins YadA and Inv facilitate binding to host cell β1-integrins^12,13. Y. pestis, however, lacks these adhesins and such interaction would not dictate preferential association of the plague bacillus with phagocytic leukocytes. In addition, elucidating the basis for the differential Yop targeting in vitro versus in vivo will be important, because such information will help us understand virulence mechanisms for other human pathogens with similar type III secretion systems, including Salmonella species and Escherichia coli^2,5.

As with most animal models, the question of how well those used by Marketon et al. and Overheim et al. represent human infections and/or immune responses remains open. Although Y. pestis infection in laboratory mice is a relatively good model of septicemic plague in humans, there are significant differences in the innate immune systems of each species.

Another major unresolved issue is the markedly increased virulence of Y. pestis compared to the other pathogenic Yersinia species. These include Y. pseudotuberculosis and Y. enterocolitica, which are relatively infrequent causes of gastrointestinal disease in humans². Unlike Y. pestis, these enteropathogens cause self-limited infections that are successfully contained in the lymph node, even though the type III secretion system and Yop armamentarium of all three species are virtually identical. It will also be important to compare Y. pestis immune evasion in animals having differing natural susceptibility to plague, and in bubonic and pneumonic plague transmission models.

The advances described by Marketon et al. and Overheim et al. highlight the importance of in vivo studies in addressing these questions, and will ultimately lead to an enhanced understanding of plague pathogenesis and vaccinology.