Host-pathogen Studies Research Articles

Novel Fosfomycin Resistance Mechanism in Pseudomonas entomophila Due to Atypical Pho Regulon Control of GlpT

Background/Objectives: Pseudomonas entomophila is a ubiquitous bacterium capable of killing insects of different orders and has become a model for host–pathogen studies and a promising tool for biological pest control. In the human pathogen Pseudomonas aeruginosa, spontaneous resistance to fosfomycin arises almost exclusively from mutations in the glycerol-3-phosphate transporter (GlpT), the drug’s sole entry route in this species. Here, we investigated whether this specificity is conserved in P. entomophila, as it could provide a valuable marker system for studying mutation rates and spectra and for selection in genetic engineering. Methods: We isolated 16 independent spontaneous fosfomycin-resistant mutants in P. entomophila, and studied the genetic basis of the resistance using a combination of sequencing, phenotyping and computational approaches. Results: We only found two mutants without alterations in glpT or any of its known regulatory elements. Whole-genome sequencing revealed unique inactivating mutations in phoU, a key regulator of the phosphate starvation (Pho) regulon. Computational analyses identified a PhoB binding site in the glpT promoter, and experiments showed that phoU inactivation reduced glpT expression nearly 20-fold. While placing a sugar-phosphate transporter under the Pho regulon may seem advantageous, bioinformatic analysis shows this configuration is atypical among pseudomonads. Conclusions: this atypical Pho regulon control of GlpT probably reflects the peculiarities of P. entomophila’s habitat and lifestyle; highlighting how readily regulatory evolution can lead to the rapid divergence of resistance mechanisms, even among closely related species.

Signals of positive selection in genomes of palearctic Myotis-bats coexisting with a fungal pathogen

Disease can act as a driving force in shaping genetic makeup across populations, even species, if the impacts influence a particularly sensitive part of their life cycles. White-nose disease is caused by a fungal pathogen infecting bats during hibernation. The mycosis has caused massive population declines of susceptible species in North America, particularly in the genus Myotis. However, Myotis bats appear to tolerate infection in Eurasia, where the fungal pathogen has co-evolved with its bat hosts for an extended period of time. Therefore, with susceptible and tolerant populations, the fungal disease provides a unique opportunity to tease apart factors contributing to tolerance at a genomic level to and gain an understanding of the evolution of non-harmful in host-parasite interactions. To investigate if the fungal disease has caused adaptation on a genomic level in Eurasian bat species, we adopted both whole-genome sequencing approaches and a literature search to compile a set of 300 genes from which to investigate signals of positive selection in genomes of 11 Eurasian bats at the codon-level. Our results indicate significant positive selection in 38 genes, many of which have a marked role in responses to infection. Our findings suggest that white-nose syndrome may have applied a significant selective pressure on Eurasian Myotis-bats in the past, which can contribute their survival in co-existence with the pathogen. Our findings provide an insight on the selective pressure pathogens afflict on their hosts using methodology that can be adapted to other host-pathogen study systems.

Ovine placental explants: A new ex vivo model to study host‒pathogen interactions in reproductive pathogens

Reproductive failure is one of the main performance constraints in ruminant livestock. Transmissible agents such as Toxoplasma gondii and Neospora caninum are commonly involved in the occurrence of abortion in ruminants, but little is known about the mechanisms involved. While in vivo models are optimal for the study of abortion pathogenesis, they have a high economic cost and come with ethical concerns. Unfortunately, alternative in vitro models fail to replicate the complex in vivo placental structure. To overcome the limitations of currently available models, we developed an ex vivo model based on the cultivation of fresh and cryopreserved sheep placental explants, enabling the biobanking of tissues. Reproducible and simple markers of tissue integrity (histology, RNA concentrations), viability (resazurin reduction), and functionality (synthesis of steroid hormones) were also investigated, allowing a clear quality assessment of the model. This work shows that, similar to fresh explants, tissues cryopreserved in ethylene glycol using slow freezing rates maintain not only their structure and function but also their receptivity to T. gondii and N. caninum infection. In addition, the findings demonstrate that explant lifespan is mainly limited by the culture method, with protocols requiring improvements to extend it beyond 2 days. These findings suggest that cryopreserved tissues can be exploited to study the initial host‒pathogen interactions taking place in the placenta, thus deepening the knowledge of the specific mechanisms that trigger reproductive failure in sheep. Importantly, this work paves the way for the development of similar models in related species and contributes to the reduction of experimental animal use in the future.

Cell scientist to watch – Géraldine Laloux

ABSTRACT Géraldine Laloux is an Associate Professor of bacterial cell biology at the de Duve Institute, UCLouvain, Belgium. After studying host–pathogen interactions during her PhD at the University of Namur, Belgium, Géraldine focused more on the cell biology of bacteria when she joined the Jacobs-Wagner lab at Yale University, USA. In 2017, she then returned to Belgium to set up her lab at UCLouvain, where she works with predatory bacteria to study their unusual cell cycle mechanisms and underlying cellular organisation. We caught up with Géraldine over Zoom to find out more about the interesting lifestyle of these predatory bacteria, as well as her approaches to mentoring and starting a new lab.

Efficient Prediction of Blast Disease in Paddy Plant using Optimized Support Vector Machine

Diagnostics of plant diseases are essential to predicting yield loss, detecting infections, studying host–pathogen interactions, and detecting host resistance. Early detection of plant leaf disease can increase yields. This is possible if there are automated systems to help farmers diagnose rice diseases from the pictures of plant leaves. Therefore, automatic leaf detection using machine learning is proposed in this study. The proposed approach has three stages, namely, pre-processing, feature extraction, and classification. Initially, the input image is converted into red, green and blue and the noise in the green band is removed using a median filter. Initially, the input image is converted into red, green and blue and the noise in the green band is removed using a median filter. Then, important features of the green band are extracted. An Optimized Support Vector Machine (OSVM) classifier uses the extracted features to classify an image as normal or pathological. To improve SVM performance, the SVM parameters are chosen optimally using the Adaptive Sunflower Optimization (ASFO) algorithm. Then, the infected region is separated using a level-set segmentation algorithm. The efficiency of our work is analyzed based on accuracy, sensitivity, and specificity, the proposed method reached the maximum accuracy of 97.54% for plant disease prediction.

Extraction of High-Quality RNA from S. aureus Internalized by Endothelial Cells.

Staphylococcus aureus evades antibiotic therapy and antimicrobial defenses by entering human host cells. Bacterial transcriptomic analysis represents an invaluable tool to unravel the complex interplay between host and pathogen. Therefore, the extraction of high-quality RNA from intracellular S. aureus lays the foundation to acquire meaningful gene expression data. In this study, we present a novel and straightforward strategy to isolate RNA from internalized S. aureus after 90 min, 24 h, and 48 h postinfection. Real-time PCR data were obtained for the target genes agrA and fnba, which play major roles during infection. The commonly used reference genes gyrB, aroE, tmRNA, gmk, and hu were analyzed under different conditions: bacteria from culture (condition I), intracellular bacteria (condition II), and across both conditions I and II. The most stable reference genes were used for the normalization of agrA and fnbA. Delta Cq (quantification cycle) values had a relatively low variability and thus demonstrated the high quality of the extracted RNA from intracellular S. aureus during the early phase of infection. The established protocol allows the extraction and purification of intracellular staphylococcal RNA while minimizing the amount of host RNA in the sample. This approach can leverage reproducible gene expression data to study host-pathogen interactions.

Infectious disease ecology and evolution in a changing world.

Infectious disease ecology and evolution in a changing world.

Localized Infections with P. aeruginosa Strains Defective in Zinc Uptake Reveal That Zebrafish Embryos Recapitulate Nutritional Immunity Responses of Higher Eukaryotes.

The innate immune responses of mammals to microbial infections include strategies based on manipulating the local concentration of metals such as iron (Fe) and zinc (Zn), commonly described as nutritional immunity. To evaluate whether these strategies are also present in zebrafish embryos, we have conducted a series of heart cavity-localized infection experiments with Pseudomonas aeruginosa strains characterized by a different ability to acquire Zn. We have found that, 48 h after infection, the bacterial strains lacking critical components of the Zn importers ZnuABC and ZrmABCD have a reduced colonization capacity compared to the wild-type strain. This observation, together with the finding of a high level of expression of Zur-regulated genes, suggests the existence of antimicrobial mechanisms based on Zn sequestration. However, we have observed that strains lacking such Zn importers have a selective advantage over the wild-type strain in the early stages of infection. Analysis of the expression of the gene that encodes for a Zn efflux pump has revealed that at short times after infection, P. aeruginosa is exposed to high concentrations of Zn. At the same time, zebrafish respond to the infection by activating the expression of the Zn transporters Slc30a1 and Slc30a4, whose mammalian homologs mediate a redistribution of Zn in phagocytes aimed at intoxicating bacteria with a metal excess. These observations indicate that teleosts share similar nutritional immunity mechanisms with higher vertebrates, and confirm the usefulness of the zebrafish model for studying host-pathogen interactions.

3D Oral Mucosal Models for Studying Host-Pathogen Interactions in Periodontal Disease

The host immune response to the oral biofilm plays a central role in the aetiology of periodontitis. Our understanding of hostpathogen interactions has historically been reliant on monolayer culture systems stimulated by microorganisms. However, 2D culture systems possess major shortcomings as these do not adequately mimic the complexities of the native tissue and therefore fail to provide meaningful information on the pathogenesis of periodontal disease. To mimic the in vivo condition, various types of 3D oral mucosal equivalents (OME) have been employed, including reconstituted models and 3D organotypic culture models. The rationale for OME is to serve as a relevant in vitro tool to examine the interactions of human epithelial cells with bacterial biofilms, to understand the process of epithelial layer damage, molecular mechanisms underpinning the development of periodontitis and models for testing novel therapeutics. Furthermore, to gain a deeper understanding of periodontal dysbiosis, it is important to appreciate the molecular mechanisms underlying host epithelial cell production of cytokines and chemokines in response to bacterial stimulation; and signalling pathways which underpin host-pathogen interactions such as mitogen-activated protein kinases (MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NFkB). Thus, the aim of this narrative review is to summarise the evidence regarding the application of 3D oral mucosal models in host-pathogen infection studies relating to periodontal disease, review the molecular mechanisms underlying host response and provide recommendations for future studies in this field.

Generation of self-replicating airway organoids from the cave nectar bat Eonycteris spelaea as a model system for studying host–pathogen interactions in the bat airway epithelium

ABSTRACT Bats are reservoir hosts for various zoonotic viruses with pandemdic potential in humans and livestock. In vitro systems for studying bat host–pathogen interactions are of significant interest. Here, we establish protocols to generate bat airway organoids (AOs) and airway epithelial cells differentiated at the air–liquid interface (ALI-AECs) from tracheal tissues of the cave-nectar bat Eonycteris spelaea. In particular, we describe steps which enable laboratories that do not have access to live bats to perform extended experimental work upon procuring an initial batch of bat primary airway tissue. Complete mucociliary differentiation required treatment with IL-13. E. spelaea ALI-AECs supported productive infection with PRV3M, an orthoreovirus for which Pteropodid bats are considered the reservoir species. However, these ALI-AECs did not support SARS-CoV-2 infection, despite E. spelaea ACE2 receptor being capable of mediating SARS-CoV-2 spike pseudovirus entry. This work provides critical model systems for assessing bat species-specific virus susceptibility and the reservoir likelihood for emerging infectious agents.

Cryptococcus neoformans releases proteins during intracellular residence that affect the outcome of the fungal-macrophage interaction.

Cryptococcus neoformans is a facultative intracellular pathogen that can replicate and disseminate in mammalian macrophages. In this study, we analyzed fungal proteins identified in murine macrophage-like cells after infection with C. neoformans. To accomplish this, we developed a protocol to identify proteins released from cryptococcal cells inside macrophage-like cells; we identified 127 proteins of fungal origin in infected macrophage-like cells. Among the proteins identified was urease, a known virulence factor, and others such as transaldolase and phospholipase D, which have catalytic activities that could contribute to virulence. This method provides a straightforward methodology to study host–pathogen interactions. We chose to study further Yeast Oligomycin Resistance (Yor1), a relatively uncharacterized protein belonging to the large family of ATP binding cassette transporter (ABC transporters). These transporters belong to a large and ancient protein family found in all extant phyla. While ABC transporters have an enormous diversity of functions across varied species, in pathogenic fungi they are better studied as drug efflux pumps. Analysis of C. neoformans yor1Δ strains revealed defects in nonlytic exocytosis, capsule size, and dimensions of extracellular vesicles, when compared to wild-type strains. We detected no difference in growth rates and cell body size. Our results indicate that C. neoformans releases a large suite of proteins during macrophage infection, some of which can modulate fungal virulence and are likely to affect the fungal–macrophage interaction.

Bombyx mori ferritin heavy-chain homolog facilitates BmNPV proliferation by inhibiting reactive oxygen species–mediated apoptosis

Ferritin heavy-chain homolog (FerHCH), an iron-binding protein, plays an important role in the host defense against oxidative stress and pathogen infections. In our previous research, Bombyx mori native ferritin had an interaction with B. mori nucleopolyhedrovirus (BmNPV). However, the underlying molecular mechanism of single ferritin homolog responses to BmNPV infection remains unclear. In this study, we found that BmNPV titer and B. mori FerHCH (BmFerHCH) expression were positively correlated with the ferric iron concentration. We performed RNA interference (RNAi) and overexpression experiments to investigate the effects of BmFerHCH on BmNPV proliferation. BmFerHCH knockdown suppressed BmNPV proliferation in vivo and in vitro, whereas BmFerHCH overexpression facilitated BmNPV proliferation. In addition, the oxidative stress level was increased significantly in BmN cells after budded virus infection, while BmFerHCH could neutralize the increased ROS production induced by BmNPV. Of note, we found that ROS was involved in BmNPV-induced apoptosis. Through inhibiting ROS, apoptosis was suppressed by BmFerHCH, whereas BmFerHCH knockdown facilitated apoptosis. Therefore, we hypothesize that BmFerHCH-mediated inhibition of virus-induced apoptosis depends on suppressing ROS accumulation and, thereby, facilitates virus replication. These results suggest that BmFerHCH plays an important role in facilitating BmNPV proliferation and modulating BmFerHCH is potential strategy for studying host–pathogen interactions.

Apical-out airway organoids as a platform for studying viral infections and screening for antiviral drugs

Airway organoids are polarized 3D epithelial structures that recapitulate the organization and many of the key functions of the in vivo tissue. They present an attractive model that can overcome some of the limitations of traditional 2D and Air–Liquid Interface (ALI) models, yet the limited accessibility of the organoids’ apical side has hindered their applications in studies focusing on host–pathogen interactions. Here, we describe a scalable, fast and efficient way to generate airway organoids with the apical side externally exposed. These apical-out airway organoids are generated in an Extracellular Matrix (ECM)-free environment from 2D-expanded bronchial epithelial cells and differentiated in suspension to develop uniformly-sized organoid cultures with robust ciliogenesis. Differentiated apical-out airway organoids are susceptible to infection with common respiratory viruses and show varying responses upon treatment with antivirals. In addition to the ease of apical accessibility, these apical-out airway organoids offer an alternative in vitro model to study host–pathogen interactions in higher throughput than the traditional air–liquid interface model.

Using Dictyostelium discoideum as a model for phagocytosis resistance in Escherichia coli

Abstract Escherichia coli related illnesses continue to be a problem around the world, especially in developing countries that lack clean water and proper sanitation. Dictyostelium discoideum is a soil dwelling amoeba that phagocytizes bacteria for nutrients. D. discoideum is a great model to study host pathogen interactions, and it has been used to identify virulence factors in bacteria. Our objective is to use Dictyostelium discoideum as a model to identify bacterial genes involved in resistance to phagocytosis. We hypothesize that D. discoideum can be used as a model organism to identify virulence genes associated with E. coli. The aims of this study are to identify E. coli genes predicted to grant resistance to D. discoideum grazing and phagocytosis and further characterize genes identified as potential virulence factors and observe the genes’ effects on D. discoideum grazing and phagocytic killing. Intestinal pathogenic, extraintestinal pathogenic, and laboratory E. coli strains were classified according to grazing phenotype. Previous comparative genomic analysis by large-scale BLAST score ratio (LS-BSR) identified genes prevalent amongst strains resistant to D. discoideum grazing. The gene gipA (growth in Peyer’s patches) was one gene found in various E. coli strains resistant to D. discoideum grazing. We are currently working to characterize the role of E. coli genes associated with resistance to D. discoideum grazing by manipulating E. coli strains for expression of these genes. We will observe the interactions between these strains and D. discoideum grazing and phagocytosis. This experiment will give insight into bacterial genes and virulence factors responsible for evading phagocytosis and the immune system. This project was funded in part by federal funds from the National Institute of Allergy and Infectious Diseases, National Institute of Health, and Department of Health and Human Services under grant number U19 AI110820. Dr. M. Snyder was funded by an NIH NIGMS grant F33 GM116618, a Franklin Research Grant from the American Philosophical Society and a grant from the TU FDRC committee. This research was also supported by an NIH NIGMS R25 GM19970.

An eco-epidemiological modeling approach to investigate dilution effect in two different tick-borne pathosystems.

Disease (re)emergence appears to be driven by biodiversity decline and environmental change. As a result, it is increasingly important to study host–pathogen interactions within the context of their ecology and evolution. The dilution effect is the concept that higher biodiversity decreases pathogen transmission. It has been observed especially in zoonotic vector‐borne pathosystems, yet evidence against it has been found. In particular, it is still debated how the community (dis)assembly assumptions and the degree of generalism of vectors and pathogens affect the direction of the biodiversity–pathogen transmission relationship. The aim of this study was to use empirical data and mechanistic models to investigate dilution mechanisms in two rodent–tick–pathogen systems differing in their vector degree of generalism. A community was assembled to include ecological interactions that expand from purely additive to purely substitutive. Such systems are excellent candidates to analyze the link between vector ecology, community (dis)assembly dynamics, and pathogen transmission. To base our mechanistic models on empirical data, rodent live‐trapping, including tick sampling, was conducted in Wales across two seasons for three consecutive years. We have developed a deterministic single‐vector, multi‐host compartmental model that includes ecological relationships with non‐host species, uniquely integrating theoretical and observational approaches. To describe pathogen transmission across a gradient of community diversity, the model was populated with parameters describing five different scenarios differing in ecological complexity; each based around one of the pathosystems: Ixodes ricinus (generalist tick)–Borrelia burgdorferi and I. trianguliceps (small mammals specialist tick)–Babesia microti. The results suggested that community composition and interspecific dynamics affected pathogen transmission with different dilution outcomes depending on the vector degree of generalism. The model provides evidence that dilution and amplification effects are not mutually exclusive in the same community but depend on vector ecology and the epidemiological output considered (i.e., the “risk” of interest). In our scenarios, more functionally diverse communities resulted in fewer infectious rodents, supporting the dilution effect. In the pathosystem with generalist vector we identified a hump shaped relationship between diversity and infections in hosts, while for that characterized by specialist tick, this relationship was more complex and more dependent upon specific parameter values.

Genetic Analysis of TB Susceptibility Variants in Ghana Reveals Candidate Protective Loci in SORBS2 and SCL11A1 Genes.

Despite advancements made toward diagnostics, tuberculosis caused by Mycobacterium africanum (Maf) and Mycobacterium tuberculosis sensu stricto (Mtbss) remains a major public health issue. Human host factors are key players in tuberculosis (TB) outcomes and treatment. Research is required to probe the interplay between host and bacterial genomes. Here, we explored the association between selected human/host genomic variants and TB disease in Ghana. Paired host genotype datum and infecting bacterial isolate information were analyzed for associations using a multinomial logistic regression. Mycobacterium tuberculosis complex (MTBC) isolates were obtained from 191 TB patients and genotyped into different phylogenetic lineages by standard methods. Two hundred and thirty-five (235) nondisease participants were used as healthy controls. A selection of 29 SNPs from TB disease-associated genes with high frequency among African populations was assayed using a TaqMan® SNP Genotyping Assay and iPLEX Gold Sequenom Mass Genotyping Array. Using 26 high-quality SNPs across 326 case-control samples in an association analysis, we found a protective variant, rs955263, in the SORBS2 gene against both Maf and Mtb infections (P BH = 0.05; OR = 0.33; 95% CI = 0.32–0.34). A relatively uncommon variant, rs17235409 in the SLC11A1 gene was observed with an even stronger protective effect against Mtb infection (MAF = 0.06; PBH = 0.04; OR = 0.05; 95% CI = 0.04–0.05). These findings suggest SLC11A1 and SORBS2 as a potential protective gene of substantial interest for TB, which is an important pathogen in West Africa, and highlight the need for in-depth host-pathogen studies in West Africa.

Label-Free Quantitative Proteomic Profiling of Fusarium Head Blight in Wheat.

To distinguish protein abundance changes in biological systems under different conditions, mass spectrometry-based proteomics provides a powerful tool to detect and quantify such responses. Improvements in mass spectrometry instrumentation sensitivity and resolution, along with advanced bioinformatics enable new strategies to study host-pathogen interactions. This protocol uses the state-of-the-art MS-based proteomics to assess infection of the global fungal pathogen Fusarium graminearum, on the world-wide cereal crop Triticum aestivum, resulting in the devastating disease of Fusarium head blight (FHB). Here, host infection is mimicked by inoculating F. graminearum onto T. aestivum cultivars (e.g., FHB-resistant and -susceptible) in the growth room under controlled environment, followed by sample harvesting at different time points (e.g., 24 and 120h post-inoculation) to assess temporal responses to infection. The collected samples are processed using our in-house pipeline for total protein extraction and quantified via label-free methods by liquid-chromatography-coupled with tandem MS/MS. From this experiment, we define dual perspectives of infection considering dynamic protein abundance changes in both the pathogen and host simultaneously, allowing us to identify strategies used by the pathogen to evade the host defense responses and those used by the host to protect from severe infection.

Reprogrammed Pteropus Bat Stem Cells as A Model to Study Host-Pathogen Interaction during Henipavirus Infection.

Bats are natural hosts for numerous zoonotic viruses, including henipaviruses, which are highly pathogenic for humans, livestock, and other mammals but do not induce clinical disease in bats. Pteropus bats are identified as a reservoir of henipaviruses and the source of transmission of the infection to humans over the past 20 years. A better understanding of the molecular and cellular mechanisms allowing bats to control viral infections requires the development of relevant, stable, and permissive cellular experimental models. By applying a somatic reprogramming protocol to Pteropus bat primary cells, using a combination of ESRRB (Estrogen Related Receptor Beta), CDX2 (Caudal type Homeobox 2), and c-MYC (MYC proto-oncogene) transcription factors, we generated bat reprogrammed cells. These cells exhibit stem cell-like characteristics and neural stem cell molecular signature. In contrast to primary fibroblastic cells, these reprogrammed stem cells are highly permissive to henipaviruses and exhibit specific transcriptomic profiles with the particular expression of certain susceptibility factors such as interferon-stimulated genes (ISG), which may be related to viral infection. These Pteropus bat reprogrammed stem cells should represent an important experimental tool to decipher interactions during henipaviruses infection in Pteropus bats, facilitate isolation and production of bat-borne viruses, and to better understand the bat biology.

Galleria mellonella as a Good Model to Study Acinetobacter baumannii Pathogenesis.

The invertebrate model, Galleria mellonella, has been widely used to study host–pathogen interactions due to its cheapness, ease of handling, and similar mammalian innate immune system. G. mellonella larvae have been proven to be useful and a reliable model for analyzing pathogenesis mechanisms of multidrug resistant Acinetobacter baumannii, an opportunistic pathogen difficult to kill. This review describes the detailed experimental design of G. mellonella/A. baumannii models, and provides a comprehensive comparison of various virulence factors and therapy strategies using the G. mellonella host. These investigations highlight the importance of this host–pathogen model for in vivo pathogen virulence studies. On the long term, further development of the G. mellonella/A. baumannii model will offer promising insights for clinical treatments of A. baumannii infection.

Targeted RNA-Seq Reveals the M. tuberculosis Transcriptome from an In Vivo Infection Model

Simple SummaryHigh-throughput sequencing techniques such as RNA-seq allow a more detailed characterization of the gene expression profile during in vivo infections. However, using this strategy for intracellular pathogens such as Mycobacterium tuberculosis (Mtb) entails technical limitations. Some authors have resorted to flow cytometers to separate infected cells or significantly increase sequencing depth to obtain pathogens’ gene expression. However, these options carry additional expenses in specialized equipment. We propose an experimental protocol based on differential cell lysis and a probe-based ribosomal depletion to determine the gene expression of Mtb and its host during in vivo infection. This method allowed us to increase the number of observed expressed genes from 13 using a traditional RNA-seq approach to 702. In addition, we observed the expression of genes essential for establishing the infection, codifying proteins such as PE-PGRS, lipoproteins lppN and LpqH, and three ncRNAs (small RNA MTS2823, transfer-messenger RNA RF00023, and ribozyme RF00010). We believe our method represents a valuable alternative to current RNA-seq approaches to study host–pathogen interactions and will help explore host–pathogen mechanisms in tuberculosis and other similar models of intracellular infections. The study of host-pathogen interactions using in vivo models with intracellular pathogens like Mycobacterium tuberculosis (Mtb) entails technical limitations, such as: (i) Selecting an efficient differential lysis system to enrich the pathogen cells; (ii) obtaining sufficient high-quality RNA; and (iii) achieving an efficient rRNA depletion. Thus, some authors had used flow cytometers to separate infected cells or significantly increase the sequencing depth of host–pathogen RNA libraries to observe the pathogens’ gene expression. However, these options carry additional expenses in specialized equipment typically not available for all laboratories. Here, we propose an experimental protocol involving differential cell lysis and a probe-based ribosomal depletion to determine the gene expression of Mtb and its host during in vivo infection. This method increased the number of observed pathogen-expressed genes from 13 using the traditional RNA-seq approach to 702. After eliminating rRNA reads, we observed that 61.59% of Mtb sequences represented 702 genes, while 38.41% represented intergenic regions. Some of the most expressed genes codified for IS1081 (Rv2512c) transposase and eight PE-PGRS members, such as PGRS49 and PGRS50. As expected, a critical percent of the expressed genes codified for secreted proteins essential for infection, such as PE68, lppN, and LpqH. Moreover, three Mtb ncRNAs were highly expressed (small RNA MTS2823, transfer-messenger RNA RF00023, and ribozyme RF00010). Many of the host-expressed genes were related to the inflammation process and the expression of surfactant proteins such as the Sftpa and Sftpc, known to bind Mtb to alveolar macrophages and mi638, a microRNA with no previous associations with pulmonary diseases. The main objective of this study is to present the method, and a general catalog of the Mtb expressed genes at one point of the in vivo infection. We believe our method represents a different approach to the existing ones to study host–pathogen interactions in tuberculosis and other similar intracellular infections, without the necessity of specialized equipment.