Negative correlation between ACE2 gene expression levels and loss of taste in a cohort of COVID-19 hospitalized patients: New clues to long-term cognitive disorders.

In early 2020, one of the most prevalent symptoms of SARS-CoV-2 infection was the loss of smell (anosmia), found in 60-70% of all cases. Anosmia used to occur early, concomitantly with other symptoms, and often persisted after recovery for an extended period, sometimes for months. In addition to smell disturbance, COVID-19 has also been associated with loss of taste (ageusia). The latest research suggests that SARS-CoV-2 could spread from the respiratory system to the brain through receptors in sustentacular cells localized to the olfactory epithelium. The virus invades human cells via the obligatory receptor, angiotensin-converting enzyme II (ACE2), and a priming protease, TMPRSS2, facilitating viral penetration. There is an abundant expression of both ACE2 and TMPRSS2 in sustentacular cells. In this study, we evaluated 102 COVID-19 hospitalized patients, of which 17.60% presented anosmia and 9.80% ageusia. ACE1, ACE2, and TMPRSS2 gene expression levels in nasopharyngeal tissue were obtained by RT-qPCR and measured using ΔCT analysis. ACE1 Alu287bp association was also evaluated. Logistic regression models were generated to estimate the effects of variables on ageusia and anosmia Association of ACE2 expression levels with ageusia. was observed (OR: 1.35; 95% CI: 1.098-1.775); however, no association was observed between TMPRSS2 and ACE1 expression levels and ageusia. No association was observed among the three genes and anosmia, and the Alu287bp polymorphism was not associated with any of the outcomes. Lastly, we discuss whetherthere is a bridge linking these initial symptoms, including molecular factors, to long-term COVID-19 health consequences such as cognitive dysfunctions.

In vitro and in vivo Characterization of Host-Pathogen Interactions of the L3881 Candida albicans Clinical Isolate.

Candida albicans is a human commensal fungus and the etiologic agent of nosocomial infections in immunocompromised individuals. Candida spp. is the most studied human fungal pathogen, and the mechanisms by which this fungus can evade the immune system affecting immunosuppressed individuals have been extensively studied. Most of these studies focus on different species of Candida, and there is much to be understood in virulence variability among lineages, specifically different C. albicans clinical isolates. To better understand the main mechanisms of its virulence variability modulated in C. albicans clinical isolates, we characterized L3881 lineage, which has been previously classified as hypovirulent, and SC5314 lineage, a virulent wild-type control, by using both in vitro and in vivo assays. Our findings demonstrated that L3881 presented higher capacity to avoid macrophage phagocytosis and higher resistance to oxidative stress than the wild type. These characteristics prevented higher mortality rates for L3881 in the animal model of candidiasis. Conversely, L3881 has been able to induce an upregulation of pro-inflammatory mediators both in vitro and in vivo. These results indicated that in vitro and in vivo functional characterizations are necessary for determination of virulence in different clinical isolates due to its modulation in the host-pathogen interactions.

Mixed signals - how Trypanosoma cruzi exploits host-cell communication and signaling to establish infection.

Chagas disease (American trypanosomiasis) is a 'neglected' pathology that affects millions of people worldwide, mainly in Latin America. Trypanosoma cruzi, the causative agent, is an obligate intracellular parasite with a complex and diverse biology that infects several mammalian species, including humans. Because of genetic variability among strains and the presence of four biochemically and morphologically distinct parasite forms, the outcome of T. cruzi infection varies considerably depending on host cell type and parasite strain. During the initial contact, cellular communication is established by host-recognition-mediated responses, followed by parasite adherence and penetration. For this purpose, T. cruzi expresses a variety of proteins that modify the host cell, enabling it to safely reach the cytoplasm. After entry into the host cell, T. cruzi forms a transitory structure termed 'parasitophorous vacuole' (PV), followed by its cytoplasmic replication and differentiation after PV rupture, and subsequent invasion of other cells. The success of infection, maintenance and survival inside host cells is facilitated by the ability of T. cruzi to subvert various host signaling mechanisms. We focus in this Review on the various mechanisms that induce host cytoskeletal rearrangements, activation of autophagy-related proteins and crosstalk among major immune response regulators, as well as recent studies on the JAK-STAT pathway.

Comparative Analysis of the Secretome and Interactome of Trypanosoma cruzi and Trypanosoma rangeli Reveals Species Specific Immune Response Modulating Proteins.

Chagas disease, a zoonosis caused by the flagellate protozoan Trypanosoma cruzi, is a chronic and systemic parasitic infection that affects ~5-7 million people worldwide, mainly in Latin America. Chagas disease is an emerging public health problem due to the lack of vaccines and effective treatments. According to recent studies, several T. cruzi secreted proteins interact with the human host during cell invasion. Moreover, some comparative studies with T. rangeli, which is non-pathogenic in humans, have been performed to identify proteins directly involved in the pathogenesis of the disease. In this study, we present an integrated analysis of canonical putative secreted proteins (PSPs) from both species. Additionally, we propose an interactome with human host and gene family clusters, and a phylogenetic inference of a selected protein. In total, we identified 322 exclusively PSPs in T. cruzi and 202 in T. rangeli. Among the PSPs identified in T. cruzi, we found several trans-sialidases, mucins, MASPs, proteins with phospholipase 2 domains (PLA2-like), and proteins with Hsp70 domains (Hsp70-like) which have been previously characterized and demonstrated to be related to T. cruzi virulence. PSPs found in T. rangeli were related to protozoan metabolism, specifically carboxylases and phosphatases. Furthermore, we also identified PSPs that may interact with the human immune system, including heat shock and MASP proteins, but in a lower number compared to T. cruzi. Interestingly, we describe a hypothetical hybrid interactome of PSPs which reveals that T. cruzi secreted molecules may be down-regulating IL-17 whilst T. rangeli may enhance the production of IL-15. These results will pave the way for a better understanding of the pathophysiology of Chagas disease and may ultimately lead to the identification of molecular targets, such as key PSPs, that could be used to minimize the health outcomes of Chagas disease by modulating the immune response triggered by T. cruzi infection.

Epithelial Haven and Autophagy Breakout in Gonococci Infection.

The World Health Organization (WHO) has estimated that in 2016, there were 87 million new cases of gonorrhea. Gonorrhea is caused by the sexually transmitted human-exclusive agent Neisseria gonorrhoeae, a Gram-negative diplococcus that causes cervicitis in females and urethritis in males and may lead to more severe complications. Currently, there is no vaccine against N. gonorrhoeae. Its resistance to antibiotics has been increasing in the past few years, reducing the range of treatment options. N. gonorrhoeae requires a surface protein/receptor (Opa proteins, porin, Type IV pili, LOS) to adhere to and invade epithelial cells. During invasion and transcytosis, N. gonorrhoeae is targeted by the autophagy pathway, a cellular maintenance process which balances sources of energy at critical times by degrading damaged organelles and macromolecules in the lysosome. Autophagy is an important host defense mechanism which targets invading pathogens. Based on transmission electron microscopy (TEM) analysis, the intracellular bacteria occupy the autophagosome, a double-membraned vesicle that is formed around molecules or microorganisms during macroautophagy and fuses with lysosomes for degradation. Most of the gonococci end up in autolysosomes for degradation, but a subpopulation of the intracellular bacteria inhibits the maturation of the autophagosome and its fusion with lysosomes by activating mTORC1 (a known suppressor of the autophagy signaling), thus escaping autophagic elimination. This mini review focuses on the cellular features of N. gonorrhoeae during epithelial cell invasion, with a particular focus on how N. gonorrhoeae evades the autophagy pathway.

The Parasitic Intracellular Lifestyle of Trypanosomatids: Parasitophorous Vacuole Development and Survival.

The trypanosomatid (protozoan) parasites Trypanosoma cruzi and Leishmania spp. are causative agents of Chagas disease and Leishmaniasis, respectively. They display high morphological plasticity, are capable of developing in both invertebrate and vertebrate hosts, and are the only trypanosomatids that can survive and multiply inside mammalian host cells. During internalization by host cells, these parasites are lodged in "parasitophorous vacuoles" (PVs) comprised of host cell endolysosomal system components. PVs effectively shelter parasites within the host cell. PV development and maturation (acidification, acquisition of membrane markers, and/or volumetric expansion) precede parasite escape from the vacuole and ultimately from the host cell, which are key determinants of infective burden and persistence. PV biogenesis varies, depending on trypanosomatid species, in terms of morphology (e.g., size), biochemical composition, and parasite-mediated processes that coopt host cell machinery. PVs play essential roles in the intracellular development (i.e., morphological differentiation and/or multiplication) of T. cruzi and Leishmania spp. They are of great research interest as potential gateways for drug delivery systems and other therapeutic strategies for suppression of parasite multiplication and control of the large spectrum of diseases caused by these trypanosomatids. This mini-review focuses on mechanisms of PV biogenesis, and processes whereby PVs of T. cruzi and Leishmania spp. promote parasite persistence within and dissemination among mammalian host cells.

Human B cells infected by Trypanosoma cruzi undergo F-actin disruption and cell death via caspase-7 activation and cleavage of phospholipase Cγ1.

B cells contribute to the immune system in many ways such as antigen presentation to CD4+ T cells, secretion of cytokines and lymphoid tissue organogenesis. Furthermore, they are the only cell type capable of producing immunoglobulins. B cells also account for critical aspects of the resistance against intracellular pathogens. Trypanosoma cruzi is an intracellular parasite that sabotages humoral response by depletion of immature B cells. Polyclonal activation and secretion of non-specific antibodies are also other mechanisms used by T cruzi to evade and subvert the mammalian host immune system, leading to increased parasitemia and susceptibility to Chagas' disease. It remained unclear whether B cell depletion occurs due to direct contact with T. cruzi or results from a global increase in inflammation. Unlike previous reports, we demonstrated in this study that T. cruzi infects human B cells, resulting in parasite-induced activation of caspase-7 followed by proteolytic cleavage of phospholipase Cγ1 and cell death. These data contribute to explain the mechanisms ruling B-cell depletion and evasion of the immune response by T. cruzi.

Human B cells infected by Trypanosoma cruzi undergo F-actin disruption and cell death via caspase-7 activation and cleavage of phospholipase Cgama1

B cells contribute to the immune system in many ways such as antigen presentation to CD4+ T cells, secretion of cytokines and lymphoid tissue organogenesis. Furthermore, they are the only cell type capable of producing immunoglobulins. B cells also account for critical aspects of the resistance against intracellular pathogens. Trypanosoma cruzi is an intracellular parasite that sabotages humoral response by depletion of immature B cells. Polyclonal activation and secretion of non-specific antibodies are also other mechanisms used by T cruzi to evade and subvert the mammalian host immune system, leading to increased parasitemia and susceptibility to Chagas’ disease. It remained unclear whether B cell depletion occurs due to direct contact with T. cruzi or results from a global increase in inflammation. Unlike previous reports, we demonstrated in this study that T. cruzi infects human B cells, resulting in parasite-induced activation of caspase-7 followed by proteolytic cleavage of phospholipase Cgama1 and cell death. These data contribute to explain the mechanisms ruling B-cell depletion and evasion of the immune response by T. cruzi.

Human B cells infected by Trypanosoma cruzi undergo F-actin disruption and cell death via caspase-7 activation and cleavage of phospholipase Cgama1

B cells contribute to the immune system in many ways such as antigen presentation to CD4+ T cells, secretion of cytokines and lymphoid tissue organogenesis. Furthermore, they are the only cell type capable of producing immunoglobulins. B cells also account for critical aspects of the resistance against intracellular pathogens. Trypanosoma cruzi is an intracellular parasite that sabotages humoral response by depletion of immature B cells. Polyclonal activation and secretion of non-specific antibodies are also other mechanisms used by T cruzi to evade and subvert the mammalian host immune system, leading to increased parasitemia and susceptibility to Chagas’ disease. It remained unclear whether B cell depletion occurs due to direct contact with T. cruzi or results from a global increase in inflammation. Unlike previous reports, we demonstrated in this study that T. cruzi infects human B cells, resulting in parasite-induced activation of caspase-7 followed by proteolytic cleavage of phospholipase Cgama1 and cell death. These data contribute to explain the mechanisms ruling B-cell depletion and evasion of the immune response by T. cruzi.

Trypanosoma cruzi extracellular amastigotes selectively trigger the PI3K/Akt and Erk pathways during HeLa cell invasion.

Cell invasion by Trypanosoma cruzi extracellular amastigotes involves different signaling pathways to induce phagocytosis-like mechanisms. Previous works indicated that PI3K/Akt, Src and Erk might be involved in EA invasion; however, participation of these molecules in this process remains elusive. Here, we observed that EA activated Akt, Erk but not Src. Interference of EA invasion with specific inhibitors corroborated this observation. Our results show that EA is capable of selectively triggering complex signaling pathways. Activation of PI3K/Akt and Erk, kinases related to actin cytoskeleton rearrangement and phagocytosis, reinforces the idea that T. cruzi EA subverts the phagocytic machinery during invasion.

Molecular Characterization of Trypanosoma evansi Mevalonate Kinase (TeMVK).

The mevalonate pathway is an essential part of isoprenoid biosynthesis leading to production of a diverse class of >30,000 biomolecules including cholesterol, heme, and all steroid hormones. In trypanosomatids, the mevalonate pathway also generates dolichols, which play an essential role in construction of glycosylphosphatidylinositol (GPI) molecules that anchor variable surface proteins (VSGs) to the plasma membrane. Isoprenoid biosynthesis involves one of the most highly regulated enzymes in nature, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), which catalyzes the conversion of HMG-CoA to mevalonic acid. The enzyme mevalonate kinase (MVK) subsequently converts mevalonic acid to 5-phosphomevalonic acid. Trypanosoma evansi is a flagellate protozoan parasite that causes the disease "Surra" in domesticated large mammals, with great economic impact. T. evansi has only a trypomastigote bloodstream form and requires constant modification of the variant surface glycoprotein (VSG) coat for protection against the host immune system. We identified MVK of T. evansi (termed TeMVK) and performed a preliminary characterization at molecular, biochemical, and cellular levels. TeMVK from parasite extract displayed molecular weight ~36 kDa, colocalized with aldolase (a glycosomal marker enzyme) in glycosomes, and is structurally similar to Leishmania major MVK. Interestingly, the active form of TeMVK is the tetrameric oligomer form, in contrast to other MVKs in which the dimeric form is active. Despite lacking organized mitochondria, T. evansi synthesizes both HMGCR transcripts and protein. Both MVK and HMGCR are expressed in T. evansi during the course of infection in animals, and therefore are potential targets for therapeutic drug design.

Candida albicans: The Ability to Invade Epithelial Cells and Survive under Oxidative Stress Is Unlinked to Hyphal Length.

In its hyphal form, Candida albicans invades epithelial and endothelial cells by two distinct mechanisms: active penetration and induced endocytosis. The latter is dependent on a reorganization of the host cytoskeleton (actin/cortactin recruitment), whilst active penetration does not rely on the host's cellular machinery. The first obstacle for the fungus to reach deep tissues is the epithelial barrier and this interaction is crucial for commensal growth, fungal pathogenicity and host defense. This study aimed to characterize in vitro epithelial HeLa cell invasion by four different isolates of C. albicans with distinct clinical backgrounds, including a C. albicans SC5314 reference strain. All isolates invaded HeLa cells, recruited actin and cortactin, and induced the phosphorylation of both Src-family kinases (SFK) and cortactin. Curiously, L3881 isolated from blood culture of a patient exhibited the highest resistance to oxidative stress, although this isolate showed reduced hyphal length and displayed the lowest cell damage and invasion rates. Collectively, these data suggest that the ability of C. albicans to invade HeLa cells, and to reach and adapt to the host's blood, including resistance to oxidative stress, may be independent of hyphal length.

Leishmania (Viannia) braziliensis Inositol Phosphorylceramide: Distinctive Sphingoid Base Composition.

Inositol phosphorylceramide (IPC), the major sphingolipid in the genus Leishmania but not found in mammals, is considered a potentially useful target for chemotherapy against leishmaniasis. Leishmania (Viannia) braziliensis is endemic in Latin America and causes American tegumentary leishmaniasis. We demonstrated that IPCs are localized internally in parasites, using a specific monoclonal antibody. Treatment with 5 µM myriocin (a serine palmitoyltransferase inhibitor) rendered promastigotes 8-fold less infective than controls in experimental hamster infection, as determined by number of parasites per inguinal lymph node after 8 weeks infection, suggesting the importance of parasite IPC or sphingolipid derivatives in parasite infectivity or survival in the host. IPC was isolated from promastigotes of three L. (V.) braziliensis strains and analyzed by positive- and negative-ion ESI-MS. The major IPC ions were characterized as eicosasphinganine and eicosasphingosine. Negative-ion ESI-MS revealed IPC ion species at m/z 778.6 (d20:1/14:0), 780.6 (d20:0/14:0), 796.6 (t20:0/14:0), 806.6 (d20:1/16:0), and 808.6 (d20:0/16:0). IPCs isolated from L. (V.) braziliensis and L. (L.) major showed significant differences in IPC ceramide composition. The major IPC ion from L. (L.) major, detected in negative-ion ESI-MS at m/z 780.6, was composed of ceramide d16:1/18:0. Our results suggest that sphingosine synthase (also known as serine palmitoyltransferase; SPT) in L. (V.) braziliensis is responsible for synthesis of a long-chain base of 20 carbons (d20), whereas SPT in L. (L.) major synthesizes a 16-carbon long-chain base (d16). A phylogenetic tree based on SPT proteins was constructed by analysis of sequence homologies in species of the Leishmania and Viannia subgenera. Results indicate that SPT gene position in L. (V.) braziliensis is completely separated from that of members of subgenus Leishmania, including L. (L.) major, L. (L.) infantum, and L. (L.) mexicana. Our findings clearly demonstrate sphingoid base differences between L. (V.) braziliensis and members of subgenus Leishmania, and are relevant to future development of more effective targeted anti-leishmaniasis drugs.

Unique behavior of Trypanosoma cruzi mevalonate kinase: A conserved glycosomal enzyme involved in host cell invasion and signaling.

Mevalonate kinase (MVK) is an essential enzyme acting in early steps of sterol isoprenoids biosynthesis, such as cholesterol in humans or ergosterol in trypanosomatids. MVK is conserved from bacteria to mammals, and localizes to glycosomes in trypanosomatids. During the course of T. cruzi MVK characterization, we found that, in addition to glycosomes, this enzyme may be secreted and modulate cell invasion. To evaluate the role of TcMVK in parasite-host cell interactions, TcMVK recombinant protein was produced and anti-TcMVK antibodies were raised in mice. TcMVK protein was detected in the supernatant of cultures of metacyclic trypomastigotes (MTs) and extracellular amastigotes (EAs) by Western blot analysis, confirming its secretion into extracellular medium. Recombinant TcMVK bound in a non-saturable dose-dependent manner to HeLa cells and positively modulated internalization of T. cruzi EAs but inhibited invasion by MTs. In HeLa cells, TcMVK induced phosphorylation of MAPK pathway components and proteins related to actin cytoskeleton modifications. We hypothesized that TcMVK is a bifunctional enzyme that in addition to playing a classical role in isoprenoid synthesis in glycosomes, it is secreted and may modulate host cell signaling required for T. cruzi invasion.

Interactions between Trypanosoma cruzi Secreted Proteins and Host Cell Signaling Pathways.

Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6-7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here, we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion.

The Mitogen-Activated Protein Kinase (MAPK) Pathway: Role in Immune Evasion by Trypanosomatids.

Leishmania spp. and Trypanosoma cruzi are the causative agents of leishmaniasis and Chagas disease, respectively, two neglected tropical diseases that affect about 25 million people worldwide. These parasites belong to the family Trypanosomatidae, and are both obligate intracellular parasites that manipulate host signaling pathways and the innate immune system to establish infection. Mitogen-activated protein kinases (MAPKs) are serine and threonine protein kinases that are highly conserved in eukaryotes, and are involved in signal transduction pathways that modulate physiological and pathophysiological cell responses. This mini-review highlights existing knowledge concerning the mechanisms that Leishmania spp. and T. cruzi have evolved to target the host's MAPK signaling pathways and highjack the immune response, and, in this manner, promote parasite maintenance in the host.

Trypanosoma cruzi extracellular amastigotes trigger the protein kinase D1-cortactin-actin pathway during cell invasion.

Trypanosoma cruzi extracellular amastigotes (EAs) display unique mechanisms for cell invasion that are highly dependent on host actin filaments. Protein kinase D1 (PKD1) phosphorylates and modulates the activity of cortactin, a key regulator of actin dynamics. We evaluated the role of host cortactin and PKD1 in actin filament dynamics during HeLa cell invasion by EAs. Host cortactin, PKD1 and actin are recruited by EAs based on experiments in fixed and live cells by time lapse confocal microscopy. EAs trigger PKD1 and extracellular signal-regulated kinase 1/2 activation, but not Src family kinases, and selectively phosphorylate cortactin. Heat-killed EAs and non-infective epimastigotes both triggered distinct host responses and did not recruit the molecules studied herein. EA invasion was influenced by depletion or overexpression of host cortactin and PKD1, respectively, suggesting the involvement of both proteins in this event. Collectively, these results show new host cell mechanisms subverted during EA internalization into non-phagocytic cells.

Genome of the avirulent human-infective trypanosome--Trypanosoma rangeli.

BACKGROUND: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts. METHODOLOGY/PRINCIPAL FINDINGS: The T. rangeli haploid genome is ∼ 24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heat-shock proteins. CONCLUSIONS/SIGNIFICANCE: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets.