Intracellular Pathogens II: Rickettsiales

Rickettsial diseases have had a profound impact on human lives for centuries but were poorly understood prior to the beginning of the 20th century. The study of the pathogens is challenging and requires an intimate understanding of arthropod vectors, mammalian hosts, and human nature. This chapter focuses on rickettsial infections of public health importance to humans, primarily focusing on rickettsiae in the United States. It also briefly addresses the global importance of rickettsial pathogens. An understanding of the epidemiology of rickettsial diseases requires not only a look at contemporary public health issues, but also an understanding of the history of these infections and classic approaches to control and prevention. Since the early investigations of Ricketts and others into the origins and ecology of Rocky Mountain spotted fever (RMSF), public health interest in rickettsiae has remained strong in the United States. The chapter concentrates on the historical and current epidemiology and public health importance of rickettsial pathogens known to occur in the United States. R. parkeri infection can be difficult to distinguish from infection with Rickettsia rickettsii, as extensive serologic cross-reactivity occurs between this and other spotted fever group rickettsial (SFGR) species. From 2000 to 2007, 3,126 cases of presumed Ehrlichia chaffeensis and an additional 824 cases classified as "Ehrlichiosis Undetermined/Unspecified/Other Agent (UUOA)" were reported within the United States. Most of these Ehrlichiosis UUOA cases were probably E. chaffeensis or Anaplasma phagocytophilum cases that could not be properly classified.

The majority of species belonging to the genus Rickettsia are divided into two groups, the spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR), based on the diseases that they cause, the presence of major surface antigens, and the ability to promote intracellular actin-based motility. This chapter focuses on the cell biology involved in the internalization of SFGR into nonphagocytic mammalian cells using Rickettsia conorii as a model organism. It highlights the current knowledge regarding the bacterial proteins and cognate host cell receptors involved in initiating this process. Early studies on the mechanism(s) utilized by rickettsiae to invade nonphagocytic mammalian cells identified cellular actin dynamics as playing an important role. In some invasive pathogens, depletion of membrane cholesterol using methyl-β-cyclodextrin disrupts the composition of lipid rafts and inhibits R.conorii invasion of nonphagocytic cells, suggesting that the presence of Ku70 within these microdomains is important for efficient bacterial entry. Mechanistic similarities to the invasion pathways utilized by zippering pathogens suggested that R.conorii also usurps these types of signaling events. Bioinformatic analyses of sequenced rickettsial genomes revealed the presence of a gene family. Four genes in this family, namely sca0, sca1, sca2, and sca5, are present as intact open reading frames in the genomes of the majority of SFGR. The signals involved in rickettsial outer membrane protein B (rompB)-dependent invasion closely resemble those observed during the invasion of R.conorii into Vero cells and include the activation of actin and microtubule dynamics and the stimulation of protein tyrosine kinase and PI-kinase activities.

Studies of representative members, primarily Anaplasma phagocytophilum and Ehrlichia chaffeensis, have shed much light on the exquisite mechanisms by which Anaplasmataceae pathogens manipulate their host cells, and are discussed following overviews of the diseases that they cause, their notable genomic features, and their infection and developmental cycles. The secretion of type IV secretion system (T4SS) effectors into host cells is critical for survival of facultative and obligate intracellular bacterial pathogens. Apoptosis is initiated by enzymatic caspases, which are inactive until they are activated by apoptotic signaling pathways. The A. phagocytophilum-occupied vacuole (ApV) excludes fusion with secretory vesicles and specific granules harboring NADPH oxidase and proteolytic enzymes. Preferentially recruiting Rab GTPases that are predominantly found on slow recycling endosomes potentially provides A. phagocytophilum with four intracellular survival advantages. First, A. phagocytophilum is auxotrophic for 16 amino acids. Second, the mechanism by which A. phagocytophilum obtains LDL endocytic pathway-derived cholesterol for incorporation into its cell wall is unknown. Third, continual delivery of recycling endosomes to the ApV would conceivably provide an unlimited supply of host membrane material to allow for expansion of the AVM, which would be necessary to accommodate growing intravacuolar bacterial populations. Fourth, by coating the AVM with recycling endosome- associated Rab GTPases, the ApV camouflages itself as a recycling endosome, which is likely a means by which it protects itself from fusing with lysosomes. Finally, much of what authors know regarding Anaplasmataceae pathogen manipulation of host cell functions is derived from studies of A. phagocytophilum and E. chaffeensis.

Although spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR) represent two major antigenically defined and historically well-known subdivisions of pathogenic Rickettsia species, recent in-depth characterization by neighbor-joining phylodendrogramic analysis has distributed rickettsiae into ancestral, spotted fever, typhus, and transitional subgroups. Importantly, well-established and widely accepted in vivo models of infection closely mimicking the pathogenesis of Rocky Mountain spotted fever (RMSF) and epidemic typhus in humans employ infection of susceptible mouse strains with Rickettsia conorii and R. typhi, respectively. The endothelial cell responses to R. prowazekii and signaling mechanisms that determine the interplay between the host and the unique rickettsial pathogen, which is capable of escaping immune surveillance to cause recrudescent infections, remain critically important but neglected areas of scientific inquiry. Thus, one of the major critical gaps in the understanding of rickettsial pathogenesis is the definition of the biological basis of rickettsial affinity and consequent interactions with vascular endothelium. R. rickettsii infection induces a biphasic pattern of NF-ΚB activation in cultured human endothelial cells that is characterized by an early transient activation phase and a late sustained phase. There is increasing recognition that apoptosis, a tightly regulated process of altruistic suicide, plays a central role in complex interactions between an invading pathogen and host cell defense. First recognized for their ability to impede viral replication, interferons (IFNs) play a critical role in determining host survival in response to viral infection. During bacterial infections, IFN signaling defends the host by integrating early innate immune responses with later events.

Genera belonging to the family Rickettsiaceae, Rickettsia and Orientia, include human pathogens. The diseases they cause are referred to as rickettsioses in this chapter. They share three critical factors with implications in pathogenesis and immunity: (i) they are transmitted by arthropod vectors; (ii) they are obligate intracellular bacteria that inhabit the cell cytoplasm; and (iii) the predominant target is the endothelium. The only exception is Rickettsia akari, the agent of rickettsial-pox, which predominantly infects monocytes and macrophages. The chapter emphasizes on some findings and suggests a framework for a modern conceptualization of the field of rickettsiology at the interface with immunology. The development of the adaptive immune response is conditioned by the innate immune mechanisms activated during early events of the infection. The study of the endothelium in the context of true endothelium-target infections offers new opportunities to explore the role of the endothelium in orchestrating or modifying immune responses. The study of the response to two of the most successful human vaccines in history, the yellow fever vaccine and the smallpox vaccine, is likely to yield relevant paradigms that we could use as guideposts in rickettsiology. Development of a modern vaccine against Rickettsiaor Orientia should aim at mimicking a physiological immune response in the sense that all branches of adaptive immunity should be stimulated. This implies the identification of a combination of antigens that together can stimulate protective responses mediated by CD8+ T cells, CD4+ T cells, and B cells (antibodies).

This chapter focuses on the genera Anaplasma and Ehrlichia. Typically, antigenically variable proteins are immunodominant and thus allow evasion of the predominant immune responses. The donor allele repertoire remains unchanged during antigenic variation, while the expression-site variant is lost. This method is employed by some rickettsiae in the family Anaplasmataceae and allows for lifelong persistence in the host with donor allele repertoires 10- to 100-fold smaller than those found in African trypanosomes. Earlier work has demonstrated that major surface proteins (MSP)2 and MSP3 are immunodominant, antigenically variable proteins that are instrumental in evading the host immune response. Anaplasma ovis, a small ruminant pathogen, has been shown to establish persistent infections in goats. Antigenic variation in A. phagocytophilum is effected through the homolog of A. marginale msp2. The antibody response to the MSP2 HVR is variant specific and diminishes rapidly, consistent with the idea that antigenic variation of MSP2 is responsible for persistence. Research shows that the implications of donor allele repertoires go beyond antigenic variation in the individual hosts, and that they also play critical roles in the epidemiology of pathogen strain structure and possibly host tropism.

This chapter reviews the research that has led to the first successes in genetic transformation of arthropod-borne bacteria belonging to the order Rickettsiales in the Alphaproteobacteria subdivision. Seminal results provided the very first evidence that direct genetic manipulation of rickettsiae was achievable, and that the bacteria were able to maintain and express foreign genetic sequences inserted via allelic exchange/homologous recombination under control of rickettsial or Escherichia coli promoters. This work set the stage for all subsequent research efforts aspiring to manipulate and analyze rickettsiae in a manner that is nearly commonplace in extracellular bacteria. The Himar1 transposase allele A7 has been successfully used for mutagenesis of A.phagocytophilum and fever groups of rickettsial tick symbionts have been spotted using fluorescent markers and antibiotic resistance. Selection of mutants using growth inhibitors is an indispensible strategy to recover mutants from the background of nontransformed bacteria. The most common strategy is incorporation of an antibiotic resistance gene in the transformation cassette. When working with human or animal pathogens, this is a sensitive issue, as introduction of resistance to antibiotics used to treat disease induced by the pathogen to be transformed is not encouraged. This poses a dilemma, as the most effective selection is likely achieved by using the clinically most effective antibiotics. The authors suggests that shuttle vectors would be useful for testing the function of genes that are naturally defective in certain Rickettsia spp., in complementation assays, overexpression of native or foreign genes, testing gene regulation, and other applications carried out in E.coli.