https://imcjms.com Ibrahim Medical College Journal of Medical Science https://imcjms.com/registration/journal_full_text/396 Wed, 27 Oct 2021 13:27:52 +0000 Abstract Rocky Mountain spotted fever (RMSF) is a bacterial infection caused by Rickettsia, a diverse group of small Gram-negative rod-shaped α-proteobacteria, and obligates intracellular pathogens, which are free-living in hosts' cell cytoplasm and are transmitted to humans by arthropod vectors. It is the most acute rickettsial diseases known to human, with significant death rates of over 20–30%. They are distinguished by a strictly intracellular position which has, for long, delayed their comprehensive study. This article attempts primarily to focus on the mechanisms of Rickettsia-host cell interactions and the underlying molecular pathogenesis of RMSF. IMC J Med Sci 2022; 16(1): 004. DOI: https://doi.org/10.55010/imcjms.16.010 *Correspondence: Bashiru Sani, Department of Microbiology, Federal University of Lafia, Nasarawa State, Nigeria. Email: bashmodulus@gmail.com   Introduction Rickettsia are a diverse group of small Gram-negative rod-shaped α-proteobacteria, usually 0.3 by 0.1μm and obligate intracellular pathogens, that lives free in the host cell cytoplasm, and are transmitted by arthropod vectors to humans [1]. Rickettsia species have a small size of approximately 1.1-1.5Mbp genome and gene content of 900-1,500 genes [2]. They are parasites of arthropods infecting insects and ticks (fleas and lice) [3,4], in which they are assumed to be able to be maintained in the population and can as well be transmitted vertically. In contact with the faeces or through the bites of the vectors, the parasites can infect mammals, thereby making it easy to become the source for the next lines of infected vectors [5]. The genus Rickettsia causes RMSF and Mediterranean spotted fever (MSF) by Rickettsia rickettsii and Rickettsia conorii respectively. At the same time, the typhus syndromes are made up of epidemic and endemic typhus due to infection with Rickettsia prowazekii and Rickettsia typhi respectively [6]. Rickettsial diseases have well-established reputation as critical human infectious diseases, leading to disability, deaths, and “scourge of armies” during World Wars I and II [6]. Although Rickettsia sp. have traditionally been separated into different groups, the spotted fever and typhus groups, a modern classification based on whole-genome put forward by Gillespie et al. has now categorised over 20 species of the genus Rickettsia into four groups [7], including the ancestral group that is made up of R. Canadensis and R. bellii which are affiliated with ticks. The typhus group consisting of R. Prowazekii and R. typhi which are affiliated with fleas and lice, the spotted fever group consisting of R. africae, R. heilongjiangensis, R. helvetica, R. slovaca, R. honei, R. japonica, R. aeschlimanii, R. massiliae, R. montanensis, R. parkeri, R. peacockii, R. rhipicephali, R. rickettsii, R. Sibirica and R. conorii which are affiliated with ticks and a transitional group consisting of R. felis, R. australis and R. akari which are affiliated with mites, fleas and ticks [7]. RMSF is the most critical rickettsial diseases known to human, with significant death rates of over 20–30% [6]. This article attempts to focus on the mechanisms underlying host pathogen interactions and the molecular pathogenesis of RMSF.   Rocky Mountain spotted fever Rocky Mountain spotted fever caused by R. Rickettsii, is attributed as the most critical rickettsial diseases known to human, with significant death rates of over 20–30%, unless treated with an appropriate antibiotic at the appropriate time [6]. The death rate and severity of the infection are more significant for men, especially black men, and older adults, when there is deficiency in glucose-6-phosphate dehydrogenase [8]. Despite the fact that RMSF was first identified over 100 years ago, diagnosing the disease remains difficult because a rash is not noticeable up to three days into the illness and the petechial rash does not manifest until later in the course [9]. As a potentially deadly tick-borne infection to human-kind, RMSF is an infection notifiable to the Centre for Disease Control and Prevention (CDC) in the United States of America. In the United States between 2000 and 2007, the reported annual incidence of RMSF rose from less than two to over seven cases per million people, but there is a decline in death rate in the post antibiotic era [9]. In the central and southern part of America, RMSF is continually present in various urban, coastal deep forest and suburban regions of Argentina, Brazil, Costa Rica, Colombia, Panama and Mexico [6]. Apart from RMSF, Mediterranean spotted fever caused by R. conorii is continually present in the Mediterranean basin and is considered a milder disease than the RMSF; however, it has been reported that the death rates in adults are as high as 21% [10]. Another significant characteristic of R. conorii transmission to humans is the existence of a tache-noir called 'eschar' seen at the tick bite site [4,11]. In addition, the potential of some other spotted fever species such as R. helvetica, R. aeschlimanii, R. slovaca and R. massiliae, which were believed to be non-pathogenic in nature, is also being acknowledged. Lastly, there is every likelihood that previously unsuspected arthropod vectors can transmit rickettsiae in the area that have very low prevalence of human rickettsioses, which suggests the pathogens’ exploitation of mechanisms to adjust to new ecological niches, while maintaining their virulence [12].   Contributions of genome sequencing to understanding rickettsiae The genomes of Rickettsia are greatly conserved, with the same gene content and synteny [13]. Their tiny genomes have evolved from gene decay, with plenty of non-functional genes and a high proportion of non-coding DNA. Their cytosolic niche, rich in amino acids, nucleotides and nutrients, has enabled Rickettsia to drop the genes that encode enzymes for sugar metabolism and for nucleotide, amino acid, and lipids, a feature likely to be responsible for inability to grow them in cell-free medium in the laboratory [13]. They contain proteins with 3 domains, passenger sequence, 5 autotransporters, a leader sequence that mediates transport across the cell membrane, and a transporter sequence that is inserted as a β-barrel into the outer envelope to carry the passenger sequence to the surface of the cell wall. Amid the autotransporters, outer membrane protein "OmpA" is found only in the spotted fever group, while the OmpB is found in all Rickettsia species. Sca 1, Sca2, and Sca 3 are involved in the adhesion process and exist as split genes [9,13].   Rickettsia-host cell interaction For the parasite to survive, proliferate and successfully transmit infection, the parasite needs to attach to and capture target host cells. Early study of adhesion-invasion mechanisms shows that drug-induced modifications of host cell or inactivation of rickettsiae have harmful effects on their entry into host cells, and due to the certainty that viability of the target bacteria and metabolic activity of the host cell were determined as the criteria for intracellular uptake of rickettsiae, the process was known as ‘induced phagocytosis’. The spotted fever group rickettsiae adhere to the host cell receptor Ku70 thereby employing surface protein, OmpB (they also use OmpA, Sca (surface cell antigens)1, and Sca 2 as adhesion proteins) [16]. Once the OmpB is attached to the host membrane protein Ku70, it enhances the recruitment of more cell receptor Ku70 molecules to the cell membrane, for further binding of OmpB. Ubiquitin ligase (a protein that recruits an E2 ubiquitin) is also recruited for subsequent rickettsial entry site where Ku70 is ubiquitinated, which then signal transduction phenomenon leading to the recruitment of Arp2/3 complex. A small guanidine triphosphatase (Cdc42), phosphoinositide 3-kinase, Src-family kinase, and protein tyrosine kinase activate Arp2/3, leading to phagocytosis of the adhered Rickettsia. There is a zipper-like structure formation as a consequence of cytoskeletal actin modification at the point of entry [17]. An additional rickettsial protein known as RickA (a group of proteins found in the spotted fever group but are not found in the typhus group), induces Arp2/3, as expressed on the rickettsial surface, thereby initiating polymerization of host cell actin [18,19]. The actin filament helps to push the Rickettsia to the host cell's surface, where the host cell membrane is disfigured from the outside and turned inward into the adjacent cell. As the host cell membrane is disrupted or disfigured from both outward and inward, Rickettsia can gain access into the adjoining cell without the Rickettsia being exposed to the extracellular environment. In the process, some rickettsiae are released through the inner open cavity or surface of blood vessels straight to the bloodstream [18,20]. To avoid death and phagolysosomal fusion, the parasite enters the host cell's cytosol where there is availability of amino acids, adenosine triphosphate (ATP), nutrients and nucleotides [21]. They secrete hemolysin C and phospholipase D, which helps to disrupt the phagosomal membrane thereby enabling the quick break free of the rickettsiae.   Pathogenesis The pathophysiological outcome of rickettsial infections is the increase in microvascular permeability due to the disruption of adherens junctions that involves development of inter endothelial gaps, conversion of the shape of endothelial cells from polygons to large spindles, and formation of stress fibres [22]. Based on the current belief, the mechanism of injury of Rickettsia-infected endothelial cells occurs as a result of oxidative stress, which causes lipid peroxidative damage to the host cell membranes [23]. Despite proof that suggests rickettsial infection causes oxidative stress in infected animals, it is yet to be determined how the spotted fever group rickettsioses causes other pathogenic mechanisms involving cytotoxic T cells or cytokines [24]. Once the parasite is introduced through the skin, it proliferates in the lymphatics and blood vessels. The parasite adheres to and then enters the vascular endothelium and vascular smooth muscle cells via surface exposed protein as well as rickettsial phospholipase [25,26]. Rickettsia target and proliferate inside the endothelial muscle cells of blood vessels [27,28].It then induces nuclear factorkappa B (NF-kB), which hinders apoptosis thereby mediating the production of proinflammatory cytokines like interleukin (IL)-1α, resulting in up-regulation of E-selectin [29]. This enables increase attachment of polymorphs to the vasculature. Once the endothelial cells are infected, it produces IL-6, IL-8, and monocyte chemoattractant protein 1. The pathological state of the endothelial cells gives rise to the activation of clotting factors, reduced perfusion of tissue, and the extravasation of fluids [9].A great expression of the endothelial cell injury is accompanied with increase microvascular permeability resulting in pulmonary oedema, hypotension, hypoalbuminemia, and hypovolemia [30].   Immune response Amid the most fascinating features of the pathogenesis of rickettsial infections are the host defence mechanisms. Studies carried out on murine models of spotted fever rickettsioses have recognized new mechanisms of immunity, which include cytokine- mediated activation of endothelial cell bactericidal control of intracellular infection and the role of autophagy in rickettsial killing. Once TNF-α and IFN-γ activate the murine endothelial cells, it then produces rickettsicidal nitric oxide by inducible nitric oxide synthetase [31]. Upon rickettsial infections, natural killer cells are activated and inhibit growth of rickettsiae in line with the production of IFN-γ. The cytotoxic CD8+ T cells are employed to clear off rickettsiae thereby eliminating the infected endothelial cells via activation of apoptosis determined by a perforin-mediated mechanism. Antibodies against rickettsial OmpA and OmpB stands to protect the host cells against re- infection [32,33]. However, antibodies to OmpA and OmpB proteins are not visible until the control of infection and recovery. Human endothelial cells activated by TNF-α, IFN-γ, IL-1β, including RANTES, kill intracellular rickettsiae via two bactericidal mechanisms: hydrogen peroxide production and nitric oxide production [31]. Human macrophages, a small target of rickettsial infections, eliminates intracellular rickettsiae after the activation via TNF-α, IFN-β as well as IL-1b through the production of hydrogen peroxide and tryptophan starvation of rickettsiae in line with degradation of tryptophan by indoleamine-2,3-deoxygenase [31].   Future prospect and conclusion Although RMSF was identified over 100 years ago, the mechanisms by which it escape phagosome and initiate a successful intracellular infection are yet to be fully elucidated. Diagnosing the disease still remains difficult because a rash is not visible three days into the illness and does not manifest as petechial rash until later in the course. Nevertheless, with the advances in the understanding of Rickettsia host pathogen interaction, virulence mechanisms, structures of bacterial effectors proteins, target cells, including signal transduction systems and signalling pathways, apoptotic clearance of infected cells, as well as immunopathological basis of clinical manifestation is helping in providing current target for treatment/remedy to obstruct pathogen virulence mechanism including host pathogen interaction. In recent years, molecular approaches for rickettsial disease detection and diagnosis have substantially improved diagnostic capacities. These methods are rapid and highly standardized. Combination of qPCR with eschar swabbing has allowed for more rapid and robust detection of rickettsial diseases than traditional skin biopsy. Whole-genome sequencing (WGS) has also been used to reveal unbeknownst knowledge regarding the evolutionary and physiological characteristics of rickettsiae, its proteins, secretion systems and virulence factors leading to the development of novel rickettsia detection and control strategies. It is now imperative and necessary to continuously develop cost-effective and more rapid molecular and serological diagnostic methods – especially due to extensive human migration and varying and wide range of habitats that rickettsial vector can inhabit and survive. To improve present diagnostic capacities and safeguard citizens from severe rickettsial/oriental disease, the development of such diagnostic instruments will be critical. This updated approach will be valuable not just in surveillance studies, but also in clinical circumstances where biopsies are not possible.   Conflict of interest: None.   Financial disclosure: The authors declared that this study has received no external financial support.   References 1.     Mansueto P, Vitale G, Cascio A, Seidita A, Pepe I, Carroccio A, et al. New insight into immunity and immunopathology of Rickettsial diseases. Clin Dev Immunol. 2012; 2012: 967852. 2.     Andersson SGE, Zomorodipour A, Andersson JO, Sicheritz-Pontén T, Alsmark UCM, Podowski RM, et al. The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature. 1998; 396: 133–140. 3.     Azad AF, Beard CB. Rickettsial pathogens and their arthropod vectors. Emerg Infect Dis. 1998; 4: 179–186. 4.     Raoult D, Roux V. Rickettsioses as paradigms of new or emerging infectious diseases. Clin Microbiol Rev. 1997; 10: 694–719. 5.     Blanc G, Ogata H, Robert C, Audic S, Suhre K, Vestris G, et al. Reductive genome evolution from the mother of Rickettsia. PLoS Genet. 2007; 3(1):  e14. 6.     Sahni SK, Narra HP, Sahni A, Walker DH. Recent molecular insights into rickettsial pathogenesis and immunity. Future Microbiol. 2013; 8: 1265–1288. 7.     Gillespie JJ, Beier MS, Rahman MS, Ammerman NC, Shallom JM, Purkayastha A, et al. Plasmids and Rickettsial evolution: Insight from Rickettsia felis. PLoS One. 2007; 2: e266. 8.     Walker DH. Rickettsia. In:Cockerham WC, Quah SR, editors. International Encyclopedia of Public Health, 2nd eds. Massachusetts: Academic Press; 2016; p370-377. 9.     Thorner AR, Walker DH, Petri WA. Rocky mountain spotted fever. Clin Infect Dis. 1998; 27: 1353–1360. 10.  Argüello AP, Hun L, Rivera P, Taylor L. Case report: A fatal urban case of Rocky Mountain spotted fever presenting an eschar in San José, Costa Rica. Am J Trop Med Hyg. 2012; 87:   345–348. 11.  Walker DH, Occhino C, Tringali GR, Rosa S Di, Mansueto S. Pathogenesis of rickettsial eschars: The tache noire of boutonneuse fever. Hum Pathol. 1988; 19: 1449–1454. 12.  Dumler JS, Walker DH. Rocky Mountain Spotted Fever — Changing ecology and persisting virulence. 2005; 353: 551–553. 13.  McLeod MP, Qin X, Karpathy SE, Gioia J, Highlander SK, Fox GE, et al. Complete genome sequence of Rickettsia typhi and comparison with sequences of other rickettsiae. J Bacteriol. 2004; 186: 5842–5855. 14.  Walker TS, Winkler HH. Penetration of cultured mouse fibroblasts (L cells) by Rickettsia prowazeki. Infect Immun. 1978; 22: 200–208. 15.  Walker TS. Rickettsial interactions with human endothelial cells in vitro: Adherence and entry. Infect Immun. 1984; 44: 205–210. 16.  Martinez JJ, Seveau S, Veiga E, Matsuyama S, Cossart P. Ku70, a component of DNA-dependent protein kinase, is a mammalian receptor for Rickettsia conorii. Cell. 2005; 123: 1013–1023. 17.  Martinez JJ, Cossart P. Early signaling events involved in the entry of Rickettsia conorii into mammalian cells. J Cell Sci. 2004; 117: 5097–5106. 18.  Gouin E, Egile C, Dehoux P, Villiers V, Adams J, Gertler F, et al. The RickA protein of Rickettsia conorii activates the Arp2/3 complex. Nature. 2004; 427: 457–461. 19.  Jeng RL, Goley ED, D’Alessio JA, Chaga OY, Svitkina TM, Borisy GG, et al. A Rickettsia WASP-like protein activates the Arp2/3 complex and mediates actin-based motility. Cell Microbiol. 2004; 6: 761–769. 20.  Gouin E, Gantelet H, Egile C, Lasa I, Ohayon H, Villiers V, et al. A comparative study of the actin-based motilities of the pathogenic bacteria Listeria monocytogenes, Shigella flexneri and Rickettsia conorii. J Cell Sci. 1999; 112: 1697–1708. 21.  Whitworth T, Popov VL, Yu XJ, Walker DH, Bouyer DH. Expression of the Rickettsia prowazekii pld or tlyC gene in Salmonella enterica serovar typhimurium mediates phagosomal escape. Infect Immun. 2005; 73: 6668–6673. 22.  Valbuena G, Walker DH. Changes in the adherens junctions of human endothelial cells infected with spotted fever group rickettsiae. Virchows Arch. 2005; 446: 379–382. 23.  Walker DH. Rickettsiae and rickettsial infections: The current state of knowledge. Clin Infect Dis. 2007; 45 (Supplement 1): S39–S44. 24.  Rydkina E, Sahni SK, Santucci LA, Turpin LC, Baggs RB, Silverman DJ. Selective modulation of antioxidant enzyme activities in host tissues during Rickettsia conorii infection. Microb Pathog. 2004; 36: 293–301. 25.  Silverman DJ, Santucci LA, Meyers N, Sekeyova Z. Penetration of host cells by Rickettsia rickettsii appears to be mediated by a phospholipase of rickettsial origin. Infect Immun. 1992; 60: 2733–40. 26.  Walker DH, Lane TW. Rocky Mountain spotted fever: Clinical signs, symptoms, and pathophysiology. In: Walker DH, editor. Biol. Ricketts. Dis., Boca Raton: CRC Press; 1988, p. 63–78. 27.  Walker D., Cain B. The rickettsial plaque. Evidence for direct cytopathic effect of Rickettsia rickettsii. Lab Investig. 1980; 43: 388–396. 28.  Walker DH, Firth WT, Edgell CJS. Human endothelial cell culture plaques induced by Rickettsia rickettsii. Infect Immun. 1982; 37: 301–306. 29.  Joshi SG, Francis CW, Silverman DJ, Sahni SK. NF-κB activation suppresses host cell apoptosis during Rickettsia rickettsii infection via regulatory effects on intracellular localization or levels of apoptogenic and anti-apoptotic proteins. FEMS Microbiol Lett. 2004; 234: 333–341. 30.  Walker DH, Raoult D. Rickettsia rickettsii and other spotted fever group rickettsiae (Rocky Mountain spotted fever and other spotted fevers). In: Mandell G, Bennett J, Dolin R, editors. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 6th ed. Philadelphia: Churchill Livingstone; 2005, p.729. 31.  Valbuena G, Feng HM, Walker DH. Mechanisms of immunity against rickettsiae. New perspectives and opportunities offered by unusual intracellular parasites. Microbes Infect. 2002; 4: 625–33. 32.  Feng HM, Whitworth T, Popov V, Walker DH. Effect of antibody on the Rickettsia-host cell interaction. Infect Immun. 2004; 72: 3524–3530. 33.  Feng HM, Whitworth T, Olano JP, Popov VL, Walker DH. Fc-Dependent polyclonal antibodies and antibodies to outer membrane proteins A and B, but not to lipopolysaccharide, protect SCID mice against fatal Rickettsia conorii infection. Infect Immun. 2004; 72: 2222–2228. 2026 Ibrahim Medical College. All rights reserved.