A molecular toolbox for fast and convenient diagnosis of emerging and reemerging bacterial pathogens causing fever of intermediate duration.

PURPOSE: Fever of intermediate duration (FID) is defined as a fever in the community without a specific origin or focus, with a duration between 7 and 28 days. FID is often caused by pathogens associated with animal contact or their arthropods parasites, such as ticks, fleas, or lice. The purpose of this work is to design a collection of molecular tools to promptly and accurately detect common bacterial pathogens causing FID, including bacteria belonging to genera Rickettsia, Bartonella, Anaplasma, and Ehrlichia, as well as Coxiella burnetii. METHODS: Reference DNA sequences from a collection of Rickettsia, Bartonella, Anaplasma, and Ehrlichia species were used to design genus-specific primers and FRET probes targeted to conserved genomic regions. For C. burnetii, primers previously described were used, in combination with a newly designed specific probe. Real-time PCR assays were optimized using reference bacterial genomic DNA in a background of human genomic DNA. RESULTS: The four real-time PCR assays can detect as few as ten copies of target DNA from those five genera of FDI-causing bacteria in a background of 300 ng of human genomic DNA, mimicking the low microbial load generally found in patient's blood. CONCLUSION: These assays constitute a fast and convenient "toolbox" that can be easily implemented in diagnostic laboratories to provide timely and accurate detection of bacterial pathogens that are typical etiological causes of febrile syndromes such as FID in humans.

Evolving immunometabolic response to the early Leishmania infantum infection in the spleen of BALB/c mice described by gene expression profiling.

Transcriptional analysis is a useful approximation towards the identification of global changes in host-pathogen interaction, in order to elucidate tissue-specific immune responses that drive the immunopathology of the disease. For this purpose, expression of 223 genes involved in innate and adaptive immune response, lipid metabolism, prostaglandin synthesis, C-type lectin receptors and MAPK signaling pathway, among other processes, were analyzed during the early infection in spleens of BALB/c mice infected by Leishmania infantum. Our results highlight the activation of immune responses in spleen tissue as early as 1 day p.i., but a mixed pro-inflammatory and regulatory response at day 10 p.i., failing to induce an effective response towards control of Leishmania infection in the spleen. This ineffective response is coupled to downregulation of metabolic markers relevant for pathways related to icosanoid biosynthesis, adipocytokine signaling or HIF-1 signaling, among others. Interestingly, the over-representation of processes related to immune response, revealed Il21 as a potential early biomarker of L. infantum infection in the spleen. These results provide insights into the relationships between immune and metabolic responses at transcriptional level during the first days of infection in the L. infantum-BALB/c experimental model, revealing the deregulation of many important pathways and processes crucial for parasitic control in infected tissues.

Differential expression profile of genes involved in the immune response associated to progression of chronic Chagas disease.

BACKGROUND: Patients with chronic Chagas disease present marked clinical and immunological heterogeneity. During the disease, multiple immune mechanisms are activated to fight the parasite. The purpose of this study was to investigate the expression patterns of genes involved in relevant immunological processes throughout the disease in patients with chronic Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: High-throughput RT-qPCR with QuantStudio 12K Flex real-time PCR system was used to evaluate the expression of 106 immune-related genes in PBMC from a cohort of cardiac Chagas disease patients (CCC I), asymptomatic patients (IND) and healthy donors (HD) after being stimulated with T. cruzi soluble antigens. Principal component analysis (PCA), cluster analysis and volcano plots were used to identify differentially expressed genes. In addition, gene set enrichment analysis (GSEA) was employed to identify the enriched immunological pathways in which these genes are involved. PCA revealed the existence of a statistically divergent expression profile of the 36 genes correlated with PC1 between CCC I patients and HD (p < 0.0001). Differential gene expression analysis revealed upregulation of 41 genes (expression fold-change > 1.5) and downregulation of 14 genes (expression fold-change < 0.66) (p = 8.4x10-13 to p = 0.007) in CCC I patients versus HD. Furthermore, significant differences in the expression level of specific genes have been identified between CCC I and IND patients (8 up and 1 downregulated). GSEA showed that several upregulated genes in CCC I patients participate in immunological pathways such as antigen-dependent B cell activation, stress induction of HSP regulation, NO2-dependent IL12 pathway in NK cells, cytokines-inflammatory response and IL-10 anti-inflammatory signaling. CONCLUSIONS: Cardiac Chagas disease patients show an antigen-specific differential gene expression profile in which several relevant immunological pathways seem to be activated. Assessment of gene expression profiles reveal unique insights into the immune response that occurs along chronic Chagas disease.

Gene Expression Profiling of Classically Activated Macrophages in Leishmania infantum Infection: Response to Metabolic Pre-Stimulus with Itaconic Acid.

Leishmania infection of phagocytic cells, such as macrophages, induces the differentiation of infected cells into different phenotypes according to their surrounding microenvironments. The classical activation of macrophages involves metabolic reprogramming, in which several metabolites such as succinate, fumarate and itaconate are accumulated. The immunoregulatory functions of itaconate in the context of Leishmania infection were investigated in this paper. Ex vivo bone marrow-derived macrophages were differentiated into classically activated macrophages through IFNG activation and infection with Leishmania infantum. A high-throughput real-time qPCR experiment was designed for the analyses of 223 genes involved in immune response and metabolism. The transcriptional profile of classically activated macrophages revealed the enrichment of the IFNG response pathways and the upregulation of genes such as Cxcl9, Irf1, Acod1, Il12b, Il12rb1, Nos2 or Stat1. In vitro pre-stimulation with itaconate induced a loss of the parasite control and the upregulation of genes related to local acute inflammatory response. Our results reveal that itaconate accumulation dampened classically activated macrophage antiparasitic activity, and this is reflected by the differential expression of the Il12b, Icosl and Mki67 genes. The possibility of inducing parasite-killing responses in the host through metabolic reprograming is an interesting approach for the treatment of Leishmania infections that will undoubtedly attract increasing attention in the coming years.

Study of the Etiology of Acute Respiratory Infections in Children Under 5 Years at the Dr. Agostinho Neto Hospital, Praia, Santiago Island, Cabo Verde.

Background: Acute respiratory infections are one of the major causes of morbidity and mortality in children under 5 years in developing countries and are a challenge for the health system of these countries. In Cabo Verde, despite the lack of recent studies, data indicate that it affects thousands of children, being the fourth leading cause of infant mortality in 2013. The aim of this study was to identify and describe the etiological agents associated with acute respiratory tract infections in children under 5 years old, and their associated risk factors, such as clinical symptoms or socio-demographic characteristics. Methods: Naso-pharyngeal samples were collected from children under 5 years attending at Dr. Agostinho Neto Hospital (Praia, Santiago Island, Cabo Verde) with suspected ARI at different time-points during 2019. Samples were analyzed using FilmArray® Respiratory Panel v. 2.0 Plus to identify etiological agents of ARI. A questionnaire with socio-demographic information was also collected for each participant. Data analyses were carried out using the IBM SPSS version 25 (IBM Corporation, Armonk, NY) and R 3.5.1 statistical software. Results: A total of 129 naso-pharyngeal samples were included in the study. Seventeen different etiologic agents of respiratory infections were identified. HRV/EV was the most frequent agent detected, followed by FluA H3 and RSV. Coinfection with two or more pathogens was detected in up to 20% of positive samples. The results were analyzed in terms of age-group, sex, period of the year and other social and demographic factors. Conclusion: Viruses are the main causative agents of ARI in children <5 years attending at the pediatrics service at the Dr. Agostinho Neto Hospital in Praia city, Santiago Island, Cabo Verde. Some factors are described in this study as statistically associated with the presence of an infectious agent, such as having one or more children sharing the bedroom with an adult and the presence of some clinical symptoms. The data addresses the need for studies on respiratory tract infections in Cabo Verde.

Differential Expression of Immune Response Genes in Asymptomatic Chronic Chagas Disease Patients Versus Healthy Subjects.

Infection by the Trypanosoma cruzi parasite causes Chagas disease and triggers multiple immune mechanisms in the host to combat the pathogen. Chagas disease has a variable clinical presentation and progression, producing in the chronic phase a fragile balance between the host immune response and parasite replication that keeps patients in a clinically silent asymptomatic stage for years. Since the parasite is intracellular and replicates within cells, the cell-mediated response of the host adaptive immunity plays a critical role. This function is mainly orchestrated by T lymphocytes, which recognize parasite antigens and promote specific functions to control the infection. However, little is known about the immunological markers associated with this asymptomatic stage of the disease. In this large-scale analysis, the differential expression of 106 immune system-related genes has been analyzed using high-throughput qPCR in T. cruzi antigen-stimulated PBMC from chronic Chagas disease patients with indeterminate form (IND) and healthy donors (HD) from endemic and non-endemic areas of Chagas disease. This analysis revealed that there were no differences in the expression level of most genes under study between healthy donors from endemic and non-endemic areas determined by PCA and differential gene expression analysis. Instead, PCA revealed the existence of different expression profiles between IND patients and HD (p < 0.0001), dependent on the 32 genes included in PC1. Differential gene expression analysis also revealed 23 upregulated genes (expression fold change > 2) and 11 downregulated genes (expression fold change < 0.5) in IND patients versus HD. Enrichment analysis showed that several upregulated genes in IND patients participate in relevant immunological pathways such as antigen-dependent B cell activation, stress induction of HSP regulation, NO2-dependent IL12 pathway in NK cells, and cytokine-inflammatory response. The antigen-specific differential gene expression profile detected in these patients and the relevant immunological pathways that seem to be activated could represent potential biomarkers of the asymptomatic form of Chagas disease, helpful to diagnosis and infection control.

Early Transcriptional Liver Signatures in Experimental Visceral Leishmaniasis.

Transcriptional analysis of complex biological scenarios has been used extensively, even though sometimes the results of such analysis may prove imprecise or difficult to interpret due to an overwhelming amount of information. In this study, a large-scale real-time qPCR experiment was coupled to multivariate statistical analysis in order to describe the main immunological events underlying the early L. infantum infection in livers of BALB/c mice. High-throughput qPCR was used to evaluate the expression of 223 genes related to immunological response and metabolism 1, 3, 5, and 10 days post infection. This integrative analysis showed strikingly different gene signatures at 1 and 10 days post infection, revealing the progression of infection in the experimental model based on the upregulation of particular immunological response patterns and mediators. The gene signature 1 day post infection was not only characterized by the upregulation of mediators involved in interferon signaling and cell chemotaxis, but also the upregulation of some inhibitory markers. In contrast, at 10 days post infection, the upregulation of many inflammatory and Th1 markers characterized a more defined gene signature with the upregulation of mediators in the IL-12 signaling pathway. Our results reveal a significant connection between the expression of innate immune response and metabolic and inhibitory markers in early L. infantum infection of the liver.

Transcriptional Profiling of Immune-Related Genes in Leishmania infantum-Infected Mice: Identification of Potential Biomarkers of Infection and Progression of Disease.

Leishmania spp. is a protozoan parasite that affects millions of people around the world. At present, there is no effective vaccine to prevent leishmaniases in humans. A major limitation in vaccine development is the lack of precise understanding of the particular immunological mechanisms that allow parasite survival in the host. The parasite-host cell interaction induces dramatic changes in transcriptome patterns in both organisms, therefore, a detailed analysis of gene expression in infected tissues will contribute to the evaluation of drug and vaccine candidates, the identification of potential biomarkers, and the understanding of the immunological pathways that lead to protection or progression of disease. In this large-scale analysis, differential expression of 112 immune-related genes has been analyzed using high-throughput qPCR in spleens of infected and naïve Balb/c mice at four different time points. This analysis revealed that early response against Leishmania infection is characterized by the upregulation of Th1 markers and M1-macrophage activation molecules such as Ifng, Stat1, Cxcl9, Cxcl10, Ccr5, Cxcr3, Xcl1, and Ccl3. This activation doesn't protect spleen from infection, since parasitic burden rises along time. This marked difference in gene expression between infected and control mice disappears during intermediate stages of infection, probably related to the strong anti-inflammatory and immunosuppresory signals that are activated early upon infection (Ctla4) or remain activated throughout the experiment (Il18bp). The overexpression of these Th1/M1 markers is restored later in the chronic phase (8 wpi), suggesting the generation of a classical "protective response" against leishmaniasis. Nonetheless, the parasitic burden rockets at this timepoint. This apparent contradiction can be explained by the generation of a regulatory immune response characterized by overexpression of Ifng, Tnfa, Il10, and downregulation Il4 that counteracts the Th1/M1 response. This large pool of data was also used to identify potential biomarkers of infection and parasitic burden in spleen, on the bases of two different regression models. Given the results, gene expression signature analysis appears as a useful tool to identify mechanisms involved in disease outcome and to establish a rational approach for the identification of potential biomarkers useful for monitoring disease progression, new therapies or vaccine development.

The Challenge of Stability in High-Throughput Gene Expression Analysis: Comprehensive Selection and Evaluation of Reference Genes for BALB/c Mice Spleen Samples in the Leishmania infantum Infection Model.

The interaction of Leishmania with BALB/c mice induces dramatic changes in transcriptome patterns in the parasite, but also in the target organs (spleen, liver…) due to its response against infection. Real-time quantitative PCR (qPCR) is an interesting approach to analyze these changes and understand the immunological pathways that lead to protection or progression of disease. However, qPCR results need to be normalized against one or more reference genes (RG) to correct for non-specific experimental variation. The development of technical platforms for high-throughput qPCR analysis, and powerful software for analysis of qPCR data, have acknowledged the problem that some reference genes widely used due to their known or suspected "housekeeping" roles, should be avoided due to high expression variability across different tissues or experimental conditions. In this paper we evaluated the stability of 112 genes using three different algorithms: geNorm, NormFinder and RefFinder in spleen samples from BALB/c mice under different experimental conditions (control and Leishmania infantum-infected mice). Despite minor discrepancies in the stability ranking shown by the three methods, most genes show very similar performance as RG (either good or poor) across this massive data set. Our results show that some of the genes traditionally used as RG in this model (i.e. B2m, Polr2a and Tbp) are clearly outperformed by others. In particular, the combination of Il2rg + Itgb2 was identified among the best scoring candidate RG for every group of mice and every algorithm used in this experimental model. Finally, we have demonstrated that using "traditional" vs rationally-selected RG for normalization of gene expression data may lead to loss of statistical significance of gene expression changes when using large-scale platforms, and therefore misinterpretation of results. Taken together, our results highlight the need for a comprehensive, high-throughput search for the most stable reference genes in each particular experimental model.

Characterization of monomeric DNA-binding protein Histone H1 in Leishmania braziliensis.

Histone H1 in Leishmania presents relevant differences compared to higher eukaryote counterparts, such as the lack of a DNA-binding central globular domain. Despite that, it is apparently fully functional since its differential expression levels have been related to changes in chromatin condensation and infectivity, among other features. The localization and the aggregation state of L. braziliensis H1 has been determined by immunolocalization, mass spectrometry, cross-linking and electrophoretic mobility shift assays. Analysis of H1 sequences from the Leishmania Genome Database revealed that our protein is included in a very divergent group of histones H1 that is present only in L. braziliensis. An antibody raised against recombinant L. braziliensis H1 recognized specifically that protein by immunoblot in L. braziliensis extracts, but not in other Leishmania species, a consequence of the sequence divergences observed among Leishmania species. Mass spectrometry analysis and in vitro DNA-binding experiments have also proven that L. braziliensis H1 is monomeric in solution, but oligomerizes upon binding to DNA. Finally, despite the lack of a globular domain, L. braziliensis H1 is able to form complexes with DNA in vitro, with higher affinity for supercoiled compared to linear DNA.

The tail of the ParG DNA segregation protein remodels ParF polymers and enhances ATP hydrolysis via an arginine finger-like motif.

The ParF protein of plasmid TP228 belongs to the ubiquitous superfamily of ParA ATPases that drive DNA segregation in bacteria. ATP-bound ParF polymerizes into multistranded filaments. The partner protein ParG is dimeric, consisting of C-termini that interweave into a ribbon-helix-helix domain contacting the centromeric DNA and unstructured N-termini. ParG stimulates ATP hydrolysis by ParF approximately 30-fold. Here, we establish that the mobile tails of ParG are crucial for this enhancement and that arginine R19 within the tail is absolutely required for activation of ParF nucleotide hydrolysis. R19 is part of an arginine finger-like loop in ParG that is predicted to intercalate into the ParF nucleotide-binding pocket thereby promoting ATP hydrolysis. Significantly, mutations of R19 abrogated DNA segregation in vivo, proving that intracellular stimulation of ATP hydrolysis by ParG is a key regulatory process for partitioning. Furthermore, ParG bundles ParF-ATP filaments as well as promoting nucleotide-independent polymerization. The N-terminal flexible tail is required for both activities, because N-terminal DeltaParG polypeptides are defective in both functions. Strikingly, the critical arginine finger-like residue R19 is dispensable for ParG-mediated remodeling of ParF polymers, revealing that the ParG N-terminal tail possesses two separable activities in the interplay with ParF: a catalytic function during ATP hydrolysis and a mechanical role in modulation of polymerization. We speculate that activation of nucleotide hydrolysis via an arginine finger loop may be a conserved, regulatory mechanism of ParA family members and their partner proteins, including ParA-ParB and Soj-Spo0J that mediate DNA segregation and MinD-MinE that determine septum localization.

The unstructured N-terminal tail of ParG modulates assembly of a quaternary nucleoprotein complex in transcription repression.

ParG is the prototype of a group of small (<10 kDa) proteins involved in accurate plasmid segregation. The protein is a dimeric DNA binding factor, which consists of symmetric paired C-terminal domains that interleave into a ribbon-helix-helix fold that is crucial for the interaction with DNA, and unstructured N-terminal domains of previously unknown function. Here the ParG protein is shown to be a transcriptional repressor of the parFG genes. The protein assembles on its operator site initially as a tetramer (dimer of dimers) and, at elevated protein concentrations, as a pair of tetramers. Progressive deletion of the mobile N-terminal tails concomitantly decreased transcriptional repression by ParG and perturbed the DNA binding kinetics of the protein. The flexible tails are not necessary for ParG dimerization but instead modulate the organization of a higher order nucleoprotein complex that is crucial for proper transcriptional repression. This is achieved by transient associations between the flexible and folded domains in complex with the target DNA. Numerous ParG homologs encoded by plasmids of Gram-negative bacteria similarly are predicted to possess N-terminal disordered tails, suggesting that this is a common feature of partition operon autoregulation. The results provide new insights into the role of natively unfolded domains in protein function, the molecular mechanisms of transcription regulation, and the control of plasmid segregation.

Cloning and characterization of the Leishmania (Viannia) braziliensis Hsp70 gene. Diagnostic use of the C-terminal fragment rLb70(513-663).

Cross-reactions between Leishmania braziliensis and Trypanosoma cruzi caused by common antigenic determinants hinder the specific diagnosis of cutaneous and mucocutaneous leishmaniasis (CL and MCL). Therefore, the usefulness of the 70-kDa heat shock protein (Hsp70) from L. braziliensis for differential serodiagnosis was investigated. The single-copy gene encoding Hsp70, consisting of 663 amino acids, was isolated from a genomic DNA library. The antigenicity data show that Hsp70 is an immunodominant antigen highly recognized (84%) by sera of patients with CL and MCL and to a lesser extent by chagasic patients (18.75%). Antigenic mapping of the 5 overlapping fragments into which the protein was split showed that the main antigenic determinants are located in the carboxy-terminal end. The linear antigenic determinants that show cross-reactions with chagasic sera are located in the fragment rLb70(352-518). The carboxy-terminal fragment rLb70(513-663) presents 70% sensitivity and 100% specificity, so it could be a potential candidate for specific serodiagnosis of CL and MCL caused by L. braziliensis.

Antigenicity of Leishmania braziliensis histone H1 during cutaneous leishmaniasis: localization of antigenic determinants.

The humoral immune response against Leishmania braziliensis histone H1 by patients with cutaneous leishmaniasis is described. For this purpose, the protein was purified as a recombinant protein in a prokaryotic expression system and was assayed by enzyme-linked immunosorbent assay (ELISA) with a collection of sera from patients with cutaneous leishmaniasis and Chagas' disease. The assays showed that L. braziliensis histone H1 was recognized by 66% of the serum samples from patients with leishmaniasis and by 40% of the serum samples from patients with Chagas' disease, indicating that it acts as an immunogen during cutaneous leishmaniasis. In order to locate the linear antigenic determinants of this protein, a collection of synthetic peptides covering the L. braziliensis histone H1sequence was tested by ELISA. The experiments showed that the main antigenic determinant is located in the central region of this protein. Our results show that the recombinant L. braziliensis histone H1 is recognized by a significant percentage of serum samples from patients with cutaneous leishmaniasis, but use of this protein as a tool for the diagnosis of cutaneous leishmaniasis is hampered by the cross-reaction with sera from patients with Chagas' disease.