Leishmania is an important genus from the Trypanosomatidae family that infects humans by biting sandflies from the genus Phlebotomusspp. This parasite is the leishmaniasis agent, a spectrum of the disease with various clinical manifestations. Leishmaniasis, a neglected tropical disease, is endemic in over 98 countries in different parts of the world, including Asia, Africa, the Middle East, and Central and South America. Leishmaniasis comprises three main clinical features, including cutaneous (CL), mucosal (ML), and visceral leishmaniasis (VL). This disease’s other important clinical forms are disseminated CL and Post-kala-azar Dermatitis Leishmaniasis (PKDL). Among the various clinical forms of leishmaniasis, CL is the most common, with 0.7–1.2 million cases per year in the Americas, the Mediterranean Basin, the Middle East, and Central Asia.
Effective treatment is one of the best ways to control leishmaniasis. Treatment of this complex disease depends on the type of leishmaniasis and the Leishmaniasubspecies. Overall, treatment could be considered physical, such as electrotherapy and antimoniate chemicals are considered the first-line drugs, although some studies reported treatment failure. More than 70 different species of Phlebotomus and Lutzomia spp. are responsible for transmitting leishmaniasis to humans or other animal reservoirs in the Old World and the New World, respectively. In the life cycle, Leishmaniasp. has two forms of promastigotes, inside the sandfly and amastigotes, inside the mammalian cells.
Sandflies also transmit viruses and bacteria. The interaction of resident microbiota has an important effect on the pathogenic protozoa transmitted by sandfly. Little is known about the molecular interactions of sandflies with viruses and bacteria. Leishmania RNA Virus (LRV1) exists inside the Viannia and Leishmania subgenus named LRV1 and LRV2. LRV1 in L. Viannia guyanensis and L. Viannia braziliensis directly affects the pathogenesis. However, based on our knowledge, LRV2 detected in L. major does not influence the disease output. The bacteria causing bartonellosis and Oroya fever reported from Lutzomyia sp.
For finding the interaction between the pathogens and their vectors and the resident microbes, many reports have been studied[10,11]. They upgrade our knowledge about molecular mechanisms, microbiota, and pathogens interaction inside the gut vector to help us receive critical keys to design and develop the control programs. As a control strategy, paratransgenesis is an important tool for interfering with the pathogen transmission. The crucial step in this approach is the detection of suitable microorganisms that their metabolites that could affect the transmission. The commensals bacteria of Bacillus megateriumand Brevibacterium linens are considered paratransgenic blockade for Leishmaniatransmission by P. argentipes. Also, Baccillus subtilis is stably maintained inside the gut of P. argentipes. It is reported that microbiota has a significant role in ROS suppression in the L. longipalpismidgut ROS suppression, leading to Leishmania infection. Also, the nutrition and digestion of the vector are affected by microbiota. The presence of Wiggles worthiabacteria is another example of trypanosome control.
On the other hand, the other effects of bacteria are the disruption of interactions between the pathogen-vector and the production of anti-parasite factors. One of the important aspects of the vector-parasite interaction is the competence with the colonization of the bacteria and therefore presenting a protective effect. This effect has been shown by Sant’Anna et al. in protecting L. longipalpis previously infected with L. mexicana against Serratia. It has been reported that Leishmania infection resulted in the loss of bacterial diversity during the infection in vector. Louradour et al. showed that antibiotic therapy in the vector is able to prevent growth and division of Leishmania in P. duboscqi. If antibiotics treatment is applied, the depletion of microbiota inside the gut of L. longipalpis causes impairment of L. infantum replication and infective metacyclic forms development. One of the primary outcomes from the interaction between Leishmaniaand microbiota inside the midgut sandfly is the effect on mammalian immune response.
The outcome of CL is related to the presence of local microbial flora at the site of mammalian infection. However, the molecular mechanisms of interactions among Leishmania, bacteria, and the host immune responses are unclear. The most common bacteria in CL are Staphylococcus and Streptococcus. During the blood meal of sandflies, the commensal bacteria on the host’s skin enter the gut of the vector, and also the mammalian host is exposed to microbes from the sandfly gut. Therefore, the lesion is considered a source of bacterial invasion following the superinfection.
On the other hand, the families Staphylococcaceaeand Streptococcaceae are the microbiota in the midgut of sandfly. There is a complex relationship among the sandfly, Leishmania,and commensal microbiota. Knowledge of the relationship and the related molecular mechanisms is necessary to design and develop biological control strategies against leishmaniasis. In this study, we assessed the gene expression of the most important targets, including hsp10, LACK, and gp63, from the standard strain of Leishmania major (MRHO/IR/75/ER) after exposing to S. aureusATCC 25923 and group A beta-hemolytic Streptococci (GABHS).
MATERIAL & METHODS
The Iranian standard strain of L. major (MRHO/ IR/75/ER) promastigotes was stored at the Department of Parasitology and Mycology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. It was cultured in the completed RPMI 1640 (Sigma, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS; Sigma, St. Louis, USA), 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco, Pen-Strep15140), and 50 mg/ml Ampicillin (Sigma, Germany), The culture media were incubated at 25°C and sub-cultured every 2–3 days to achieve the massive culture.
The bacterial strains used in the study were Staphylococcus aureus ATCC 25923 and GABHS, gifted by the Department of Microbiology, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. The concentration of 0.5 McFarland was used to expose with L. major (MRHO/ IR/75/ER).
In vitro assessment
The promastigotes were collected after the final stage of culture and then diluted with RPMI 1640 medium until the parasite density of 0.2 million/ml. The dilution with the volume of 100 µl was spread in each well of the culture plate. Each exposure was repeated in triplicate alongside three negative controls with no exposure. All exposures were incubated for 72 h. Finally, four groups were designed, including exposure with S. aureus, exposure with GABHS, GABHS, and S. aureus, and no exposure as the negative control. The bacterial solution was added to each well in a volume of 100 µl with a density of 0.5 McFarland.
After exposure, RNA extraction was performed using an RNA extraction kit (Vivantis, South Korea). Before extraction, the wells were washed using sterile PBS in triplicate. Then, the pellet was used for RNA extraction based on instructions of the manufacturers’ manual. The extracted RNA was assessed using agarose gel electrophoresis and spectrophotometer for the quality and quantity, respectively. The samples were stored at -70°C.
The extracted RNA was used immediately for cDNA synthesis based on the protocol of the cDNA, 2-step RT-PCR kit (Vivantis, South Korea). The samples were stored at -20°C for the next steps.
The gene expression analysis was done by SYBR Green real-time PCR. The specific primer pairs, designed in this study, were GP63-F: 5’-TT- GCTCTGGCTGGAATAGG-3’ and GP63-R: 5’- CT- CATCTGTGCGGAAGTGG-3’, LACK-F: 5- GGGTC- GTACATCAAGGTGGTGTCG-3’ and LACK-R: 5’-GTAGTCGCTGTCCACGCTGTG-3’, and HSP70- F: 5’- ACAGGCATACGTCTCTCTCGCTCTGC-3’ and HSP70-R: 5’- GTCGTGTGTGTATG TGCGTGTAG- GTCT-3’. Also, the GAPDH was used as endogenous control. Amplification was done in a volume of 20, comprising 10 µl master mix SYBR Green real-time PCR, 200 nM of each primer, and 2 µl cDNA. The temperature program comprised 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 s and annealing-extension at 6°C for 10 s. Finally, the melting curve was run. The gene expression analysis was performed using ΔΔCT by the below formula
2^ΔΔCt=(Cttarget gene of TF(AQP1)-Ctreference gene of TF(GAPDH))- (Cttarget gene of TR(AQP1)- Ctreference gene of TR(GAPDH))
The statistical analysis was done using the two-way ANOVA test to compare each gene expression of hsp70, LACK, and gp63 among the groups. The p-value < 0.05 was considered significant.
This study has been approved by the Ethical Committee from Shahid Sadoughi University of Medical Sciences, Yazd, Iran, with the code of IR.SSU.REC.1397.028.
The mean relative expressions of the studied genes, including gp63, LACK, and hsp70, were assessed and compared in all groups.
Gp63 gene expression
The gene expression of gp63 was assessed in all groups that were significantly among the groups (F3,6=3.0088, p=0.000). The gp63 gene expression was reported in the group with exposure to GABHS with the mean relative quantification of 0.54±0.03. In other words, the gene expression of gp63 in the group of Leishmania with the exposure with GABHS was 1.8-fold less than the control group with no exposure [Figure 1]. The other groups showed no gp63 gene expression.
LACK gene expression
The gene expression of LACK was assessed in all groups that had significant differences among the groups (F3,6=3.0088, p=0.000). The LACK gene was expressed in all groups except the one exposure with S. aureus. The group of L. major exposed to GABHS showed the relative quantification of 2.8±0.1 fold more than the control group. It was 1.33±0.04 for the group exposed to GABHS and S. aureus together as the mixed group [Figure 2].
Hsp70 gene expression
The gene expression of hsp70 was assessed in all groups with significant differences among the groups (F3,6=3.0088, p=0.000). The hsp70 gene was expressed just in the group exposed with GABHS with relative quantification of 5.7±0.3 fold more than the control group [Figure 3].
This study reported the gene expression of three important genes encoding a critical protein in L. major,including Lmgp63, LmLACK and Lmhsp70. We showed that the group of L. major exposed to GABHS had the gene expression of all mentioned genes. The LmLACK gene from L. major was expressed in two groups exposed to GABHS alone and GABHS with S. aureus. The Lmgp63 gene expression showed just in one group with the exposure of GABHS.
GP63 is an important protein in Leishmania sp. during infection of sandfly due to its role in the survival of amazonensis in L. longipalpis, although some controversy has been reported. It is reported that GP63 has a critical role in the attachment of L. infantum and L. braziliensis to the gut of the vector. The other study also showed that during the infection in the sandfly, gp63 overexpressed in the late forms. In the present study, gp63 gene expression was reported in the control group with the promastigotes in the late phase. The other groups showed lower expression of gp63 to the extent that the group exposed with S. aureus and the group exposed to a mixture of S. aureus and GABHS showed no expression of gp63, and the group with the exposure with GABHS showed gp63 gene expression with 1.75-fold less than the one in the control group. It seems that exposure of the parasite, with both of the bacteria studied, inhibited the gp63 gene expression. The gp63 gene encodes a zinc-dependent metalloprotease on the surface of promastigotesLeishmania parasites. This important molecule modulates the immune subversion and evasion of Leishmaniaparasite. It is well known that this protein can experience cleavage of the prominent protein tyrosine phosphates (PTPs) in the cytoplasm and result in infection in mammalian. Based on our knowledge, PTPs affect inflammatory and leishmanicidal functions. Therefore, more expression of Lmgp63 could be considered for the persistence of Leishmania infection.
The hsp70 gene encodes the heat shock protein, a chaperone, and plays an important role during transmission from vector to mammalian host and, therefore, differentiation from the promastigotes to amastigotes because of exposure to various stress, including pH, temperature, and oxidants from macrophages. In this study, we showed that the gene expression of hsp70 was increased after exposure of Leishmaniamajor to GABHS.
The LACK encoding by the LACK gene is essential for the viability of the parasite, and to establish the parasite in the host, we showed that L. major after exposure to GABDH alone or in mixture with S. aureus causes the LACK gene expression, although the gene LmLACK had more expression after exposure to GABHS alone.
After that, Leishmaniasp. enters sandflies during a blood meal from the vertebrate host; it encounters microbiota colonized in the sandfly gut. In addition, bacteria on the skin of mammalian hosts are egested by blood meal. It is reported that S. aureus is a bacterium that enters the sandfly’s gut and is colonized in the midgut. This interaction between the parasite and S. aureus activates the NLRP3 inflammasome, activating the caspase-1 caused IL-1βand IL-18 releasing. Based on our knowledge, inflammasomes from L. braziliensis, by activating IL-1β, cause exacerbation of CL in murine models.
Moreover, the microbiota inside the midgut of the sandfly has critical role in the nutrition and digestion of the vector, resulting in the innate immune pathway maturation. Also, another important aspect is the induction of the innate immune system in vertebrate hosts resulting from the interaction between parasites and microbiota inside the midgut vector. On the other hand, bacteria inside the vector’s midgut may inhibit either the growth of the pathogen or the interaction between the parasite and epithelium in the vector or even by producing anti-parasite molecules. In our study, decreasing the gene expression of the gp63 may be due to the interaction with S. aureus and GABHS. Gp63 protein is an important key to virulence, attachment of the parasite to the vector epithelium, and its defenses against the antibacterial peptides. Therefore, interaction of Leishmaniawith GABHS inside the vector midgut could be considered a biological control against leishmaniasis.
In contrast, the commensalism bacterial in the vector midgut are necessary to develop pathogen virulence. However, knowledge of the interaction mechanisms between the microbiota and the vector-borne agents can be considered a novel approach to control vector-borne diseases. Some reports regarding bacteria such as Asaia sp., Ochrobactrum intermedium, and Serratiaprevent Leishmania establishment in L. longipalpis. Deyet al. approved that co-egested of Leishmania and bacteria by L. longipalpis; the established microbes lead to activation of the animal model neutrophil inflammasome following interleukin-1b improvement to sustain the neutrophil infiltration. After antibiotic treatment of the sand fly, L. donovani infection impairs. They showed that the interaction results between the Leishmania and the vector microbiota affect the outcome of the disease. More information is necessary to reveal the interaction mechanisms between Leishmania and vector microbiota to identify the targets for designing the strategies to control leishmaniasis.
This study showed that the important genes encoding LACK, gp63, and hsp70 changed their expression after exposure to the S. aureus and GABHS. These bacteria and their products can be considered the important targets to design the strategies for controlling and eradicating vector-borne diseases.
Conflict of interest:
We thank Shahid Sadoughi University of Medical Sciences for its financial support and Mrs. MarziyehModaresSanavi’s assistance.
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