Rho GTPases as pathogen targets: Focus on curable sexually transmitted infections

Abstract

Pathogens have evolved highly specialized mechanisms to infect hosts. Several microorganisms modulate the eukaryotic cell surface to facilitate their engulfment. Once internalized, they hijack the molecular machinery of the infected cell for their own benefit. At different stages of phagocytosis, particularly during invasion, certain pathogens manipulate pathways governed by small GTPases. In this review, we focus on the role of Rho proteins on curable, sexually transmitted infections caused by Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Treponema pallidum. Despite the high, worldwide frequencies of these sexually-transmitted diseases, very little is known about the strategies developed by these microorganisms to usurp key eukaryotic proteins that control intracellular signaling and actin dynamics. Improved knowledge of these molecular mechanisms will contribute to the elucidation of how these clinically important pathogens manipulate intracellular processes and parasitize their hosts.

Introduction

Rho GTPases constitute one of the 5 distinct families, Ras, Arf, Ran, Rab and Rho, of the Ras superfamily of small GTP-binding proteins.¹ These GTPases act as molecular switches, cycling between a GDP-bound form (inactive state) and a GTP-bound form (active state). This cycle is carefully regulated by more than 60 activators (guanine nucleotide exchange factors, GEFs) and over 70 inhibitors (GTPase-activating proteins, GAPs). Multiple and diverse processes of eukaryotic cells are regulated by these GTPases, and more than 100 target/effector proteins have been described.² Ras (rat sarcoma) oncoproteins are mainly involved in the control of gene expression and cell proliferation; Arf (ADP-ribosylation factor) members participate in cargo sorting and in the formation of vesicle coats along different transport pathways; Ran (Ras-related nuclear) GTPase functions in the nucleo-cytoplasmic transport of both RNA and proteins and regulates mitotic spindle and nuclear envelope assemblies; Rab (Ras-related proteins in brain) proteins are master controllers of intracellular vesicular transport; and the Rho (Ras homologous) GTPases serve as key regulators of extracellular stimulus-mediated signaling networks that regulate actin organization, cell cycle progression and gene expression.^3,4

After the discovery of Rho as a Ras-related protein in 1985, 20 genes were subsequently identified as Rho family members, RhoA, Rac1 and Cdc42 being the best characterized.^5,6 Rho GTPases participate in many essential cellular processes; the most important ones are the regulation of the assembly and organization of the actin cytoskeleton. Additionally, they regulate cell polarity, microtubule dynamics, vesicular transport pathways, G1 cell cycle progression and a multiplicity of enzymatic activities.⁷ Interestingly, Rho GTPases also play critical roles in the interaction between pathogens and host cells by controlling innate and adaptive immune responses.^8,9

Pathogens have evolved different strategies to alter host cell functions. By modifying eukaryotic GTPases, microorganisms can manipulate a variety of cellular pathways to favor microbial colonization and/or proliferation, thereby parasitizing and even killing the host cell. Rho GTPases are involved in actin reprogramming at the underlying site of adhesion of microbes to the cell surface, in the entry or invasion of host cells, in the intracellular survival of microbes, or in the polymerization of actin to propel microorganisms into adjacent cells. A large number of bacterial virulence factors target Rho GTPases. These virulence factors can be classified into 2 distinct categories: the first group includes injected effectors, in which bacteria must be in contact with their host cells to deploy their effectors via secretion apparatuses that are similar to molecular syringes;¹⁰ and the second group includes exotoxins secreted by bacteria into their environs, which does not require direct contact with the target cell.^8,11 Once the toxin reaches the cell surface, it binds to specific receptors and enters the cell by endocytosis. Both injected effectors and exotoxins alter GTPase function through direct chemical modifications of the molecule or by interfering with regulatory elements of the GTPase cycle.

This review focuses on the relationship between Rho GTPases and 4 sexually transmitted pathogens, 3 bacteria (Chlamydia trachomatis, Neisseria gonorrhoeae, and Treponema pallidum) and the parasite Trichomonas vaginalis. A recent World Health Organization (WHO) study (http://www.who.int/reproductivehealth/publications/rtis/stisestimates/en/) estimates that 498 million adults, aged between 15 and 49, are infected with at least one of these microorganisms. Therefore, knowledge of the interplay between these pathogens and human cells is of great importance.

Chlamydia trachomatis

C. trachomatis is the major causative agent of bacterial sexually transmitted diseases and preventable blindness worldwide. Infections can result in urethritis, cervicitis, epididimitis to trachoma, lymphogranuloma venereum, pelvic inflammatory disease, tubal obstruction, ectopic pregnancy and infertility.¹² Persistent infections have recently been linked to severe chronic inflammatory diseases and cancer.¹³

C. trachomatis is a Gram-negative, obligate intracellular bacterium that is restricted to humans. It has developed diverse strategies to invade, survive and multiply within eukaryotic cells.^14,15 Chlamydia resides in a vacuole, called “inclusion,” where it avoids intracellular degradation and acquires nutrients and structural molecules from host cells.^16–19 Chlamydia displays a unique biphasic lifecycle that starts when the infectious bacterial form, the elementary body (EB), enters the cell. Then, the EB differentiates into a larger, metabolically active, but non-infectious form, the reticulate body (RB), which multiplies by binary fission. After numerous rounds of replication, RBs undergo transformation back into infectious EBs to disseminate to adjacent cells.^20–22

Rho GTPases in Chlamydia Entry

Invasion starts with the attachment of EBs to the plasma membrane of host cells. This binding is highly specific and efficient, and has been termed parasite-specific phagocytosis.²³ Despite the importance of this early event in chlamydial pathogenesis, the specific receptor-ligand interaction involved in bacterial entry remains elusive. The diversity of chlamydial strains and eukaryotic cells used in different studies, the variability in the experimental setups, and the difficulty of genetically manipulating these bacteria are the main reasons for this lack of consensus regarding the adhesins and ligands involved in Chlamydia entry.^24–28 Independently of host cell types or bacterial serovars, the unifying feature of chlamydial entry is actin remodelling at attachment sites, an event controlled by Rho GTPases.

Rho GTPases involved in the internalization step appear to be species-specific; only Rac1 is involved in C. trachomatis entry,^29,30 whereas both Cdc42 and Rac1 are activated during C. caviae invasion.³¹ Basically, Rac1, which is recruited to the entry sites where actin polymerizes,³⁰ is rapidly and transiently activated after the binding of C. trachomatis to host cells. It is likely that C. trachomatis activates a cascade involving both bacterial and host proteins, which results in the rearrangement of the actin cytoskeleton and leads to successful colonization of the host cell. In fact, after bacterial attachment, EBs secrete a protein called Translocated Actin Recruiting Protein (TARP), which is injected into the host cell cytosol through a type III secretion system (a multiprotein needle-like delivery system).^32,33 Once on the cytosolic face of the plasma membrane, TARP is phosphorylated on its N-terminal, tyrosine-rich tandem repeats by host Src³⁴ and Abelson (Abl) kinases.²⁹ This phosphorylation allows TARP to recruit the GEFs Sos1 and Vav2, which in turn activate Rac1.³⁵ Subsequently, Rac1 recruits WAVE2 and Abl interactor 1 (Abi-1), leading to actin-related protein (Arp2/3) complex activation and actin recruitment and polymerization at the bacterial binding site.³⁶ It has been proposed that a synergistic action between both bacterial and host cell proteins promotes invasion. In addition, other host tyrosine kinases, such as platelet derived growth factor receptor (PDFGR) and feline Gardner-Rasheed sarcoma viral oncogene homolog (FGR), are phosphorylated upon infection and recruited to the Chlamydia attachment site. It is possible that these kinases might function redundantly in the internalization step.^29,37 The last stage in the cascade is probably regulated by another bacterial protein, CT166, which inactivates Rac1 (but not Rho A) via glucosylation,³⁸ thus completing the activation/inactivation cycle of Rac1. In summation, this is a complex and tightly regulated process in which diverse bacterial and host proteins play essential roles in the attachment and entry of Chlamydia, in which Rac1 plays an important function throughout the process (Fig. 1A).

Figure 1.

Schematic comparison of Rho GTPases involved in Chlamydia trachomatis and Neisseria gonorrhoeae-host cell interplay. Bacterial proteins are shown in red boxes. Yellow arrows indicate Rho GTPase cycling. Phosphorylation events are shown in light blue. Putative roles of Rho GTPases are indicated in purple boxes. Bacterial exit processes are represented in green. (A) Rho GTPases and Chlamydia trachomatis. The bacterial infective form (Elementary Body, EB), through the injection of the bacterial factor TARP (Translocated Actin Recruitment Protein), triggers phosphorylation events and the recruitment of the guanine-nucleotide exchange factors (GEFs), Sos1 and Vav2, leading to Rac1 activation. Then, Rac1-GTP recruits WAVE2 and Abi-1, promoting Arp2/3 complex activation and actin polymerization at the bacterial entry site. After chlamydial differentiation into the replicative form (Reticulate Body, RB) and numerous rounds of multiplication, the Chlamydia-containing vacuole is extruded from the infected host cell by a mechanism that involves WASP, myosin II and RhoA. (B) Rho GTPases and Neisseria gonorrhoeae. Opa_CEA-expressing Neisseria gonorrhoeae (Ng), through Opa-CEACAM3 recognition, triggers Rac1 and Cdc42 activation, and the downstream recruitment of WAVE2 and the actin recruiting protein Arp2/3, which ends in actin cytoskeleton rearrangements and bacterial internalization. In contrast, Rho-mediated entry triggered by the bacterial porin PorB_IA drives actin polymerization that promotes the internalization of piliated gonococci into non-phagocytic cells. The release of Neisseria-containing phagosomes might involve Rho GTPases and actin reorganization.

Rho GTPases in Chlamydia Exit

Paradoxically, another member of the Rho family is involved in the release of the bacteria from infected cells. After their intracellular multiplication, Chlamydia disseminate and colonize neighboring cells. Two different mutually exclusive mechanisms have been described. The first one involves the lysis of the infected cells in an ordered sequence of membrane ruptures, beginning with the inclusion membrane, followed by the nuclear envelope and finally the plasma membrane. The second mechanism, known as extrusion, consists of pinching off the inclusion, protrusion out of the cell within a membrane compartment, and ultimately detachment from the host cell. The extrusion mechanism requires actin polymerization, neuronal Wiskott–Aldrich syndrome protein (WASP), myosin II and the GTPase Rho A³⁹ (Fig. 1A).

Rho GTPases at Other Stages of Chlamydial Infection

Infection-triggered arthritis

The triggering of joint inflammation by infectious agents has been well established in the case of reactive arthritis.⁴⁰ Infection with the obligate intracellular pathogen C. trachomatis is a common antecedent for reactive arthritis, as the synocyte is a suitable host cell for these bacteria.⁴¹ In this regard, Zhang et al. demonstrated that Rac proteins have a dual role in the outcome of Chlamydia-triggered arthritis.⁴² In the acute phase, Rac functions to exacerbate joint inflammation by promoting neutrophil migration, whereas during the chronic phase, Rac expression serves to alleviate arthritis, probably aiding the host clearance of Chlamydia and coordinating the appropriate expression of Toll-like receptor 4 (TLR-4) in neutrophils.⁴³

Neisseria gonorrhoeae

Neisseria gonorrhoeae (the gonococcus) is the causative agent of gonorrhoeae, which is exclusively a human disease. These bacteria colonize the mucosae of the urethra, endocervix, fallopian tube, rectum, conjunctiva and pharynx. Neisserial infections range in severity from acute infections with good prognoses to severe diseases, such as purulent arthritis, pelvic inflammatory disease, endocarditis and meningitis.⁴⁴ In addition, chronic infections can lead to ectopic pregnancies and infertility.⁴⁵

N. gonorrhoeae is a Gram-negative bacterium with a typical signature: a high capacity to modulate its antigenic surface via phase variations that result from changes in gene expression. Additionally, it rapidly accumulates point mutations, acquiring antibiotic resistance, which are the basis of its ability to colonize host cells and evade immune system surveillance.^46,47 Gonococci can reside inside several human cell types (neutrophils, monocytes, mucosal epithelial cells, endothelial cells) or act as extracellular pathogens.

Rho GTPases in Neisseria Entry

The infection begins with the attachment of the bacteria to the apical surface of mucosal cells via the gonococcal pilus.^48,49 N. gonorrhoeae approach the cell host by extending their pili, and this is followed by their retraction or disassembly. Pilus retraction⁵⁰ triggers a tight association between the bacterial opacity-associated (Opa) outer membrane proteins and the host cell receptors, which promotes invasion.^48,50–53 A single gonococcal strain can harbour 11 opa genes that can be switched on and off independently. These genes contain 5′ tandem repeats [CTCTT]n that cause high-frequency, phase-variable expression,⁵⁴ in addition to intra- and inter-genomic recombination events.^55,56 Opa adhesins are classified into 2 main groups, Opa_HS or Opa_CEA, according to their host cell receptor. The heparan-sulfate proteoglycan (HSPG) receptors present in epithelial cells interact with one particular Opa_HS variant, Opa_50,⁵⁷ while the carcinoembryonic antigen cell adhesion molecules (CEACAM) expressed by a variety of cell types recognize Opa_CEA.^58–60 The CEACAM family belongs to the immunoglobulin superfamily of adhesion molecules,⁶¹ and 4 members are receptors for Opa proteins: CEA, CEACAM1, CEACAM3 and CEACAM6.^62,63 Even when the first cell-cell contact prior to host-pathogen adherence is carried out by pili, the specificity of the binding is controlled by Opa proteins. In addition to proteoglycan-mediated adherence that involves Opa₅₀, several CEACAM receptors are differentially recognized by gonococcal Opa_CEA variants. The strategies targeting these receptors might constitute an interesting alternative for drug design against gonorrhoea, especially to overcome the antigenic variability of Neisseria and its high capacity to generate antibiotic resistance.

Rho GTPases in CEACAM-mediated neisseria entry

The involvement of Rho GTPases varies depending on the receptor used for internalization. Invasion mediated by CEACAM1 and CEACAM6 does not require actin cytoskeleton reorganization; however, entry involving CEACAM3 triggers a high and localized reorganization of the host cell surface that is regulated by Rac1 and Cdc42, but not Rho A.⁶⁴ The most distinctive feature of CEACAM3 is the presence of a sequence in its cytoplasmic domain that is reminiscent of an immunoreceptor tyrosine-based activation motif (ITAM) characterized by 2 precisely spaced tyrosine residues in a particular sequence context. Of note, CEACAM3 is exclusively expressed in neutrophils, and the majority of Opa-expressing Neisseria interact with neutrophils, leading to non-opsonic phagocytosis.^59,65,66 Recent studies indicate that the CEACAM3-ITAM sequence associates directly with Vav, a Rac GEF, thus promoting the phagocytosis and elimination of CEACAM-bound bacteria.⁶⁷ Interestingly, Vav directly associates via its Src homology 2 (SH2) domain with a phosphorylated tyrosine residue within the ITAM-like sequence of the receptor. Homologous SH2 domains are also found in the adaptor molecules Nck1 and Nck2, and mediate their interaction with CEACAM3 in a complex with the Rac effector WAVE2. Finally, these steps lead to F-actin polymerization during Neisseria uptake⁶⁸ (Fig. 1B).

Rac1 and Cdc42 also participate in the activation of proinflammatory cytokines via a cascade of cellular stress response kinases, leading to the activation of JNK/AP1 via p21-activated kinase1 (PAK),⁶⁹ in addition to their roles in bacteria internalization.^70–74

Rho GTPases in porin-mediated neisseria entry

Porins are the major component of the outer membrane of pathogenic Neisseria species. In primary cervical epithelial cells, bacterial entry involves the binding of PorB, pili and lipooligosacharide to complement receptor type 3.^75,76 In non-professional phagocytes, bacterial internalization is mediated by the PorB porin subtype A (PorB_IA).⁷⁷ PorB_IA is a bacterial GTP binding protein that forms a voltage-gated channel that translocates into mammalian cell membranes and modulates host cell signaling events. This porin-mediated mechanism of entry is independent of the Opa proteins.⁷⁷ In addition, this invasion process is not dependent on host microtubules, PI3K, or Src kinases, but requires low intracellular phosphate levels.⁷⁸ It is noteworthy that the signaling cascades triggered by PorB_IA in epithelial cells involve Rho GTPases and actin (Fig. 1B).

Rho GTPases in caveolin-mediated impairment of neisseria entry

Interestingly, type IV piliated N. gonorrhoeae induce the recruitment of host cell caveolin-1 (Cav1) and trigger Cav1 phosphorylation and downstream phosphotyrosine signaling. These events lead to cytoskeletal rearrangements that impair bacterial internalization. In brief, Cav1 interacts directly with Vav2, a Rho-family GEF, and both Vav2 and its substrate, RhoA, play a major role in the Cav1-mediated prevention of bacterial uptake.⁷⁹

Rho GTPases in Neisseria Exit

After invading the host cell through the apical region, N. gonorrhoeae traverses the cell by transcytosis, exiting through the basal region.⁸⁰ Host dynein and kinesin play a role in microtubule-mediated Neisseria transit across the cell. Conversely, myosin I is involved in the movement along the actin filament network.⁸¹ At present, the mechanisms used by this bacterium to disrupt the plasma membrane and disseminate into surrounding cells are not clear. This process may require actin reorganization, and it could be orchestrated by the activity of Rho GTPases (Fig. 1B). The exit process requires further study to unravel the molecular participants and their functions.

Rho GTPases at Other Stages of Gonococcal Infection

Rho GTPases and apoptosis

The PorB porin may induce apoptosis in epithelial cells and phagocytes.⁸² It is the only neisserial factor identified thus far that induces apoptosis in infected cells. Two host pro-apoptotic proteins, Bim and Bmf, act as crucial regulators of neisserial-induced apoptosis. Both eukaryotic proteins belong to the Bcl^-2 family, and they function synergistically. Bim and Bmf are associated with the cytoskeleton and are released in a Rac-1-dependent manner after gonococcal infection.⁸³

Trichomonas vaginalis

Trichomonas vaginalis, an extracellular protozoan parasite, is the etiological causative agent of trichomonosis, a widely disseminated, non-viral, sexually transmitted disease in humans.⁸⁴ The symptoms associated with infection in women include abdominal pain, vulvar itching, purulence, the presence of a frothy malodorous discharge with leucorrhoea, signs of colpitis macularis, and vulvar and vaginal erythema.⁸⁴ In men, it is usually asymptomatic; however, occasionally it can cause pruritus, urethritis with or without purulent secretions, epididymitis and prostatitis.⁸⁵ Additionally, T. vaginalis can cause infertility in both men and women,^84,86 and has been associated with an increased risk of acquiring HIV, cervical cancer and aggressive prostate cancer.^84,87 Despite the high incidence of trichomonosis, the exact mechanism of infection used by T. vaginalis is not well established.

Trichomonas Attachment: Participation of Rho-Related GTPases in Actin Remodeling

Despite its extracellular nature, this protozoan must adhere to epithelial cells in the urogenital tract to survive. Several adhesins have been implicated, including multifunctional metabolic proteins, lipophosphoglycans and membrane proteins.^88–92 Recent studies proposed a novel strategy for the adherence of T. vaginalis to host cells that involves the secretion of exosomes that have similar physical characteristics and protein compositions to mammalian exosomes. These exosomes promote both host-parasite and parasite-parasite interactions, and they play a role in the attachment of these protozoa to host epithelial cells.⁹³

Actually, the binding to host cells causes the transition of T. vaginalis from an ovoid, free-swimming parasite into an amoeboid, highly adherent form.⁹⁴ This process involves a transcriptomic response in the parasite that is triggered by contact with the host cell surface, which increases the expression of genes related to protein synthesis, phenotypic plasticity and host cell degradation. In fact, a major up-regulation of actin and actin-associated genes is evident, suggesting a role for the cytoskeleton in the amoeboid transition.⁹⁵ Interestingly, TvRsp, a Rac1 homolog gene, has been found in T. vaginalis.⁹⁶ The relationship between the Rac-related genes of this protozoan and actin cytoskeleton rearrangements remains to be further explored (Fig. 2).

Figure 2.

Schema depicting Rho GTPases involved in Trichomonas vaginalis-host cell interplay. Putative roles for Rho GTPases are indicated in purple boxes. Rho GTPase homologues are probably involved in the actin reorganization that occurs inside the parasite during the transition from the ovoid, free-swimming form to the amoeboid form, after its attachment to the host cell. In addition, Rho-related GTPases and actin polymerization apparently play a role in phagocytic processes that take place within the parasite.

Putative Role for Rho-Related GTPases in Nutrient Uptake by T. Vaginalis

T. vaginalis attachment to the host is important not only for establishing infection, but also for nutrient acquisition. However, a crucial source of nutrients comes from phagocytic processes occurring within these parasites. It is now clear that T. vaginalis is a phagocytic cell, able to efficiently ingest particles, bacteria and even mammalian cells.⁹⁷ In immune cells, phagocytosis involves the reorganization of the cytoskeleton and relies on the control of actin dynamics by Rho GTPases.^97,98 In contrast, the precise functions of actin and Rho-related GTPases during the phagocytic processes occurring in T. vaginalis remain elusive (Fig. 2).

Treponema pallidum

Treponema pallidum is the causative agent of syphilis, a chronic and multi-stage disease, with a diverse and wide range of clinical manifestations.⁹⁹ The lack of treatment can lead to the development of primary, secondary and tertiary syphilis. The first stage is characterized by the appearance of a single, painless lesion (the chancre) at the site of inoculation. This pathogen is sexually transmitted through microabrasions in mucosal membranes where T. pallidum adheres to epithelial cells and extracellular matrix components of the skin and mucosa. In the second stage, characterized by a disseminated maculopapular rash, T. pallidum trespasses intercellular junctions of the endothelium, resulting in haematogenous dissemination and the seeding of the central nervous system and the remainder of the body.^100–102 This pathogen may also use transcytosis to spread through the endothelium.¹⁰⁰ In the tertiary and last stage, the disease may progress to cardiovascular disease, lesions of the skin, bones or viscera (gummata), or neurosyphilis.¹⁰³

T. pallidum belongs to the Spirochaetaceae family of spiral-shaped bacteria. It is exclusively a human pathogen, able to survive for decades within the host, but extremely fragile ex vivo. This fragility represents one of the main challenges for its study. At present, it can only be transiently cultured in rabbit testes.¹⁰⁴ Analysis of the T. pallidum genome led to the identification of numerous transport proteins and a limited number of genes encoding metabolic and biosynthetic enzymes, indicating that Treponema relies heavily on scavenging compounds from the human host.¹⁰⁵

Treponema pallidum attachment to host cells

Infection starts with the attachment of T. pallidum to a wide variety of cell types, including epithelial, fibroblast-like and endothelial cells.¹⁰⁶ After binding to the epithelial surface, this microorganism traverses the tissue barrier and enters the circulation by passing through the tight junctions between endothelial cells.¹⁰⁰ T. pallidum invasion results in widespread dissemination.

The host cell components involved in Treponema attachment include host serum, cell membrane compounds, and the extracellular matrix.¹⁰⁷ In particular, host cell fibronectin has been identified as the main target for bacterial adhesion. Actually, several proteins expressed in T. pallidum have been described as adhesins. Among them, Tp0155 binds to matrix fibronectin, whereas Tp0483 binds to both the soluble and matrix forms of fibronectin of host cells.¹⁰⁸ To enter the circulatory system and penetrate cells, Tp0155 is required for attachment in the bloodstream, and Tp0483 is involved in tissue attachment; in both cases, attachment is promoted via binding to fibronectin. Tp0136 is another bacterial adhesin that binds to fibronectin and laminin, 2 host extracellular matrix glycoproteins.¹⁰⁹ In addition, Tp0750 has a dual function, as it binds specifically to a variety of laminin isoforms,¹¹⁰ and it is able to degrade host molecules, suggesting that it plays a role in the proteolytic mechanisms involved in tissue damage, destruction and dissemination of T. pallidum during infection¹¹¹ (Fig. 3).

Figure 3.

Schema depicting Rho GTPases involved in Treponema pallidum-host cell interplay. Putative roles for Rho GTPases are indicated in purple boxes. The bacterial adhesins Tp0136, Tp0155, Tp0483 and Tp0750, which binds to host fibronectin and/or laminin, are involved in the attachment to host cells. Treponema binding to cell surface might trigger actin polymerization that is regulated by Rho GTPases. The details of the events occurring at the plasma membrane are enlarged in the lower part of the panel.

Fibronectin and laminin may act in concert with Rho GTPases in a variety of physiological and pathological processes in eukaryotic cells.^112–115 In non-infected cells, Rac1, RhoA and Cdc42, in connection with fibronectin and laminin, are involved in polarization, cell cycle progression¹¹⁷ and cell-cell adhesion.¹¹⁸ Interestingly, some microorganisms, through the release of adhesion molecules, bind to fibronectin and activate Rho GTPases to promote host cell invasion.¹¹⁹ For instance, the bacterial fibronectin-binding protein of Campylobacter jejuni, CadF, and the intact flagellum are involved in eukaryotic Rac1 GTPase activation and host cell invasion.^116,120 It has been postulated that CadF binding to fibronectin results in integrin clustering and activation. These events lead to the activation of signaling cascades that involves epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK), resulting in association of c-Src and phosphorylation of paxillin. Finally, the recruitment of Dock180, a Rac1-specific guanine nucleotide exchange factor, provokes the activation of Rac1, leading to a local restructuring of the actin cytoskeleton and the engulfment of the bacteria.^116,120 Similarly, in infections caused by T. pallidum, the bacterial adhesins might target fibronectin and laminin not only to enhance the binding to host cells^108–110 but also to trigger intracellular signaling pathways that might promote Rac1 activation and consequently actin remodelling at the attachment site, facilitating the invasion of host tissues. Nevertheless, the role of Rho GTPases and the actin cytoskeleton in treponemal infections remains almost unexplored.

Conclusion

In pathogen-host cell interactions, the cytoskeleton plays a central role. It is necessary for epithelial and endothelial barrier integrity, and for preventing and limiting the invasion and dissemination of the pathogen to other tissues.¹²¹ It also participates actively in several processes in immune cells: migration,¹²² phagocytosis,⁹ immune synapsis and cell signaling.^123,124 Actin dynamics are critical for phagocytosis and endocytosis in a wide variety of cell types. Conveniently, several pathogens, through diverse mechanisms, manipulate the cytoskeleton to infect host cells.¹²⁵

Rho GTPases participate in multiple and essential cellular functions, acting as the most important controllers of the assembly and organization of the actin cytoskeleton. Therefore, it is not surprising that pathogens target Rho GTPases to promote their entry into host cells and to manipulate actin-dependent processes of infected cells. Here, we update the actual knowledge about the role played by Rho GTPases in infections caused by 4 different pathogens that cause the most important (non-viral), curable, sexually transmitted diseases, and the main data are summarized in Table 1.

Table 1.

Rho GTPases in sexually-transmitted infections. The table summarizes the main characteristics of certain sexually-transmitted pathogens, plus the role of Rho GTPases in several stages of the infectious processes. In dark gray are indicated the proteins belonging to the pathogen. nd: not determined.

Pathogen	Chlamydia trachomatis	Neisseria gonorrhoeae	Trichomonas vaginalis	Treponema pallidum
Pathogen type	Bacteria Gram (-)	Bacteria Gram (-) (diplococci)	Flagellated protozoan	Bacteria Gram (-) (spirochetae)
Niche	Intracellular obligate	Intracellular facultative	Extracellular	Extracellular
Host cell type	Epithelial cells	Epithelial cells	Epithelial cells	Epithelial, fibroblast-like and endotelial cells
Step	Entry	Exit	CEACAM-mediated entry	PorinA-mediated entry	Ovoid-ameboid transition	Attachment to host cell
Member of the Rho family	Rac1	RhoA	Rac1 – Cdc42	RhoA	nd	nd
Rho activity regulators	Vav2- Sos1 (GEFs)	nd	Vav (GEF) - Nck	nd	nd	nd
Rho effectors	WAVE2- Abi1	MyoII- WASP	WAVE2-Arp2/3	nd	nd	nd
Pathogen molecules	TARP (actin recruiting protein)	CT 166 (Rac1 inactivator)	nd	PorBIA (GTP-binding protein)	TvRSP (Rac1 homolog)	nd

C. trachomatis, a Gram-negative, obligate intracellular bacterium, utilizes Rac1 to facilitate its entry into host cells, and it uses RhoA to exit host cells and to disseminate to neighboring tissues.

N. gonorrhoeae, a Gram-negative, facultative intracellular bacterium, utilizes Rac1 and Cdc42 to promote Opa-CEACAM3 mediated entry into phagocytic host cells, and Rho for PorB_IA-mediated entry for piliated gonococci in non-phagocytic host cells. In both cases, a signaling cascade involving phosphorylation events, kinases, GTPases and GEFs leads to the final actin cytoskeleton rearrangement at the sites of entry of the bacteria.

T. vaginalis, an extracellular protozoan, does not provoke significant cytoskeletal rearrangements in host cells. Instead, actin reorganization occurs in the parasite itself. The role of Rho GTPases is not fully unravelled, but the parasite has genes that encode Rho homologues. Further investigation of T. vaginalis Rho-like GTPases and their relationship with the actin cytoskeleton of the parasite is necessary.

Finally, in the case of T. pallidum, a spiral-shaped bacterium, the role of the actin cytoskeleton and Rho GTPases during infection remains unexplored. This is largely due to the difficulties in the culturing and manipulation of Treponema, which hinder a full description of the infection process.

Knowledge of the mechanisms of invasion, proliferation and dissemination of these pathogens should contribute to more effective treatments of these sexually transmitted diseases. Rho GTPases, which are likely involved in many stages of the infection, such as the attachment, engulfment and spread of these pathogens, are molecular targets that require further studies to determine their usefulness in the control of these sexually transmitted infectious diseases.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors apologize for not mentioning all published data due to space restrictions. We thank Andrew Lindsay (Molecular Cell Biology Laboratory, School of Biochemistry and Cell Biology, Biosciences Institute, University College Cork, Cork, Ireland) and Stephanie Miserey-Lenkei (Laboratory of Molecular mechanisms of intracellular transport, CNRS UMR 144, Curie Institute, Paris, France) for critical reading of this manuscript and useful suggestions.

Funding

Our work on pathogens-host cells interaction is supported by the Bunge&Born Foundation, Sectyp-UNCuyo, Foncyt (PICT 2116 grant) and the Argentine Council of Research (CONICET).