Cloning and differential expression analyses of Cdc42 from sheep

Brucellosis caused by Brucella spp. is a worldwide zoonosis, which affects livestock, wildlife, and humans in great numbers (3). As a global epidemic, brucellosis has been reported in over 170 countries (18). Despite being endemic in many countries, the disease remains under-reported or overlooked because of underdiagnosis (5), therefore leading to important economic losses and posing a severe health threat. At present, vaccination is one of the most effective measures to reduce the prevalence of brucellosis (3). However, vaccine strain can induce antibodies that interfere with the serological tests leading to failure in discriminating vaccinated animals from naturally infected ones (3, 18). Thus, it is necessary to search for diagnostic biomarkers allowing to distinguish between vaccinated and infected animals.

Cdc42 is a small GTPase, a member of the Rho subfamily, which can act as a molecular switch to control signal transduction pathways by cycling between an inactive state (GDP-bound) and an active state (GTP-bound) (10). In GTP-binding form, Cdc42 interacts with its downstream effectors to induce diverse cellular biological actions, including lamellipodia, filopodia and stress fibre formation (20), cell polarity (4), actin polymerisation (17), transport and endocytosis (19). As a key regulator of cellular actin dynamics, Cdc42 plays an important role in host defence (10, 20). Pathogen invasion is an actin-dependent process; Cdc42 can trigger extensive rearrangements of actin cytoskeleton to prevent bacteria or viruses from invading host cells (10, 16, 17). Many viruses, such as human immunodeficiency virus type 1 (HIV-1), respiratory syncytial virus (RSV), and Ebola virus (EBOV), have evolved to hijack Cdc42 to invade host cells (17). Listeria monocytogenes can induce downregulation of host Cdc42 to promote Listeria cell-cell spread (16). Furthermore, Cdc42 regulates key signalling pathways by interacting with various effector proteins, such as NF-κB, NOD1, c-Jun N-terminal kinase (JNK), and mitogen-activated protein kinase (MAPK) pathways (9). These pathways tightly control cell proliferation, migration, differentiation, and morphogenesis. Cdc42 was reported to be overexpressed in several different cancers that contributed to tumourigenesis and cancer progression (21). Even though Cdc42 is closely associated with infectious diseases and cancers; there are no reports about the relationship between Cdc42 and brucellosis until now. In our previous study, a subtractive cDNA library of buffy coat from Brucella-infected and vaccinated sheep was constructed by suppression subtractive hybridisation (SSH), and a partial cDNA of Cdc42 gene was screened and sequenced.

In this study, we cloned Cdc42 from sheep (OaCdc42) by rapid amplification of cDNA ends (RACE), and then analysed tissue distribution and differential expression levels of OaCdc42 mRNA by RT-qPCR between infected sheep challenged with B. melitensis and sheep vaccinated with B. suis S2. Our aims were to discover potential diagnostic biomarkers to discriminate vaccinated sheep from those infected with virulent Brucella.

In this study, the full-length of OaCdc42 cDNA was reported and characterised for the first time. OaCdc42 cDNA was predicted to contain a conserved domain from Thr³ to Leu¹⁷⁷, including GAP, GEF, and GDI interaction sites (Fig. 1A). GEF, GAP, and GDI were the three major regulatory proteins for regulating the cycle between the inactive GDP-bound form and active GTP-bound form, which is a typical characteristic for Rho GTPases (15). GEFs promote the exchange of GDP for GTP to activate the GTPase, whereas GAPs and GDI both negatively regulate the cycle to inactivate the GTPases. Therefore, OaCdc42 possibly has the capacity to act as a molecular switch to control signal transduction pathways by cycling between the GDP-bound form and the GTP-bound form.

The expression profile of OaCdc42 mRNA in healthy tissues was assessed. OaCdc42 mRNA was ubiquitously expressed in all tissue samples tested, although at a different level (Fig. 4). The broad expression profile of Cdc42 had been reported in buffalo (7) and swine (11). Differential expression of OaCdc42 in different tissues indicated that it may be involved in different physiological processes, such as cell growth (12), cell motility (8), apoptosis (2). The highest levels of OaCdc42 expressed in the intestine indicated that OaCdc42 must play an important role in the intestine, which needs further study.

In our study, OaCdc42 mRNA from infected or vaccinated sheep was significantly down-regulated on day 40 post-inoculation, and then significantly up-regulated 60 days post-inoculation (Fig. 5). Although OaCdc42 mRNA expression in infected sheep showed up-regulation before day 30 post-inoculation, there was no significant difference compared to the control (P > 0.05). The results implied that OaCdc42 may be weakly activated by virulent Bm, but not by avirulent S2. This is fully in line with the “stealthy” characteristics of Brucella. Brucella is a successful intracellular pathogen. It has evolved multiple strategies to evade immune response mechanisms to establish persistent infection and replication within hosts (1). The suppression of OaCdc42 mRNA may be just one of its evolved strategies. In addition, the evidence showed that Cdc42 was activated upon cell contact by virulent B. abortus, but not by non-virulent B. abortus. The inactivation of Cdc42 significantly hampered the uptake of B. abortus, whereas the activation of Cdc42 promoted the uptake of B. abortus (6). Thus, the activation of Cdc42 is required for B. abortus internalisation. However, 40 days post-inoculation the level of OaCdc42 mRNA in infected sheep was very close to the one observed in the vaccinated sheep (Fig. 5). The reason remains elusive.

Previous studies indicated that Cdc42 could be activated by Salmonella through the type III secretion system (TTSS) to induce Salmonella internalisation into host cells. After its internalisation, the activation of Cdc42 is suppressed by SptP - the TTSS bacterial effector that is a GAP, namely, Salmonella reversibly activates Cdc42 to induce its own uptake (14). Our results showed that the expression of OaCdc42 mRNA displayed fluctuations and oscillations (Fig. 5); the possible reason was that Brucella bacteria were able to reversibly activate Cdc42 to induce its own uptake.

At present, serological testing is still the primary clinical tool to diagnose brucellosis. However, lipopolysaccharide of vaccine strain of Brucella spp. is similar to the virulent strain (5), so antibodies induced by vaccine strain can interfere with serological diagnosis. Thus, routine serological diagnosis fails to distinguish vaccinated from naturally infected animals (13). It is necessary to discover a diagnostic biomarker to distinguish vaccinated animals from those naturally infected with virulent Brucella. Our results showed that the level of OaCdc42 mRNA was significantly different between infected and vaccinated sheep (P < 0.05 or P < 0.01) on days 14, 30, and 60 post-inoculation, but there was no difference (P > 0.05) between them on day 40 post-inoculation (Fig. 5). Moreover, the expression of OaCdc42 mRNAs in infected or vaccinated sheep showed irregularity. Thus, OaCdc42 failed to distinguish vaccinated animals from the ones infected with virulent Brucella during 60 days post-inoculation. Thereby, OaCdc42 is not a good potential diagnostic biomarker for differential diagnosis of brucellosis.

In summary, the full-length of OaCdc42 cDNA was reported and characterised for the first time. OaCdc42 mRNA was widely expressed in all tested tissues, and the highest expression was detected in the intestine. The expression of OaCdc42 mRNA between vaccinated and infected sheep was significantly different (P < 0.05 or P < 0.01) on days: 14, 30, and 60 post-inoculation, but no difference (P > 0.05) was noted on day 40 post-inoculation. Moreover, the level of OaCdc42 mRNA from infected or vaccinated sheep showed irregularity. As our study has demonstrated, OaCdc42 is not a good potential diagnostic biomarker to distinguish Brucella vaccinated sheep from Brucella infected sheep.