Host-pathogen Interactions Research Articles

The chosen few: Mycobacterium tuberculosis isolates for IMPAc-TB

The three programs that make up the Immune Mechanisms of Protection Against Mycobacterium tuberculosis Centers (IMPAc-TB) had to prioritize and select strains to be leveraged for this work. The CASCADE team based at Seattle Children’s Research Institute are leveraging M.tb H37Rv, M.tb CDC1551, and M.tb SA161. The HI-IMPACT team based at Harvard T.H. Chan School of Public Health, Boston, have selected M.tb Erdman as well as a novel clinical isolate recently characterized during a longitudinal study in Peru. The PHOENIX team also based at Seattle Children’s Research Institute have selected M.tb HN878 and M.tb Erdman as their isolates of choice. Here, we describe original source isolation, genomic references, key virulence characteristics, and relevant tools that make these isolates attractive for use. The global context for M.tb lineage 2 and 4 selection is reviewed including what is known about their relative abundance and acquisition of drug resistance. Host–pathogen interactions seem driven by genomic differences on each side, and these play an important role in pathogenesis and immunity. The few M.tb strains chosen for this work do not reflect the vast genomic diversity within this species. They do, however, provide specific virulence, pathology, and growth kinetics of interest to the consortium. The strains selected should not be considered as “representative” of the growing available array of M.tb isolates, but rather tools that are being used to address key outstanding questions in the field.

Microbial community dynamics in blood, faeces and oral secretions of neotropical bats in Casanare, Colombia

Bats are known reservoirs for a wide range of pathogenic microorganisms, including viruses, bacteria, fungi, helminths, and protozoa, which can be transmitted and infect other zoonotic organisms. Various studies have utilised next-generation sequencing (NGS) to describe the pathogens associated with bats. Although most have characterised microbial communities in specific body fluids, few have analysed the composition and diversity of these microbial communities across different body fluids at the individual level. In this study, we employed two next-generation sequencing techniques: amplicon-based sequencing of the V4 hypervariable region of the 16S- and 18S-rRNA genes and viral metagenomics, to describe the prokaryotic, eukaryotic, and viral communities present in blood, faeces, and oral swab samples collected from two genera of bats (Carollia and Phyllostomus) in the department of Casanare, eastern Colombia. A total of 60 samples corresponding to the three bodily fluids were processed and analysed. The results indicated that the microbial communities across the body fluids were mainly composed of bacteria, fungi, protozoa, and various DNA and RNA viruses, showing a variability of microbial genera and species. The abundances, diversity metrics, and correlations of these microorganisms displayed patterns associated with bat genus and body fluids, suggesting that the ecological characteristics of these microbial communities may be influenced by the ecological and physiological traits of the bats. Additionally, we found similar community compositions of bacteria, some fungal genera, and viruses in the three body fluids, indicating a possible circulation of these microbes within the same bat. This could be due to microbial movement from the gut microbiota to other physiological systems or transmission via blood-feeding vectors. Furthermore, our results revealed the presence of various microbes of public health concern, including Bartonella spp., Mannheimia haemolytica, Rhodotorula spp., Piroplasmida spp., Toxoplasma gondii, Alphacoronavirus spp., and Bat circovirus. The abundance of these pathogenic microbial species across the three bodily fluids suggests potential transmission routes from bats to other organisms, which may contribute to the emergence of zoonotic disease outbreaks. These findings highlight the variability of microorganisms present within the same bat and the different pathogen-host interactions that may regulate the presence and transmission of these zoonotic microbes. Further research is required to elucidate the genomic features, ecological interactions, and biological activities of these microbial communities in bats.

Strategic genetic insights and integrated approaches for successful management of blackleg in canola/rapeseed farming

AbstractThis review describes a triumphant narrative in the battle against the devastating plant pathogen complex, Leptosphaeria maculans and L. biglobosa, and the success of the world's second‐largest oilseed crop, canola/oilseed rape. Emphasizing global collaborations, the article explores successfully mitigating this destructive disease in canola/rapeseed production across Australia, Canada and Europe. It highlights how strategies may vary between continents to adapt to specific contexts. While initial resistance (R) genes proved effective, the evolution of the pathogen under crop‐induced disease pressure led to the breakdown of these genes. Now, growers in these regions have been equipped with new tools, allowing them to make informed decisions that help to keep the disease at generally low levels. A pivotal factor in this success has been a deepened understanding of the intricate science underlying the host–pathogen interaction. Concerted efforts of individual laboratories and collaborative initiatives have played an essential role in this success, including novel methods for disease control based on extensive research that has translated into developing highly resistant varieties, enhanced pathogen monitoring, improved cultivar recommendation and integrated management strategies. The review showcases many milestone advancements, including the cloning of numerous avirulence genes within the pathogen, characterization of specific R genes, the development of various molecular tools for monitoring both pathogen and host, the introduction of groundbreaking disease management strategies such as R gene labelling, rotation and stacking, and establishment of a universal pathogen isolate collection that facilitates the exchange of information among multiple laboratories and adds a new dimension to this triumph.

Lumpy Skin Disease: A review of epidemiological study and preventive measures

Background: Lumpy skin disease (LSD) is a highly infectious and economically important transboundary disease that is rapidly spreading to the globe. The disease causes high morbidity and a low mortality rate of infection. The animals show acute or chronic illnesses depending on the immune responses of the hosts. The economic burden of LSD manifested the poor-quality hides, a drop in milk and meat production, abortion, and death. Methods: This systematic literature review was accomplished according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The literature on lumpy skin disease has been explored over the last two decades and searched keywords on online databases such as Google Scholar, PubMed, and Scopus. Both automated and manual searching tools were used to screen the articles. The literature published other than English was rejected during the screening process. Conference papers were excluded during the screening. Results: LSD is transmitted by blood-sucking arthropods and is most prevalent in summer and rainy seasons. Exotic-bred cattle and calves are highly susceptible. Although the disease has low mortality, the high mortality of the disease prevails in endemic regions in complicated cases. Calves from unvaccinated cows should be vaccinated at any age, on the other hand, the calves from vaccinated cows should be immunized at 3 months of age. Animals should be vaccinated before the risk period. Strict biosecurity, quarantine, and immunoprophylaxis can reduce the prevalence of the disease. Conclusions: Lumpy skin disease (LSD) is an acute infectious and contagious disease affecting cattle and water buffaloes. The disease causes serious economic loss due to decreased production, skin problems, and mortality in complicated cases. Further details on genetic characterization, transmission dynamics, and host-pathogen interaction should be performed to prevent the prevalence of the disease in emerging or re-emerging countries.

Genomic characterization of Pantoea dispersa A003 isolated from a clinical patient

IntroductionPantoea dispersa is a Gram-negative bacterium generally considered as a plant pathogen and rarely causes human infections. To date, there have been 13 studies that have documented clinical infections linked to P. dispersa, with a primary emphasis on the initial identification of this pathogen. The genomic features and the mechanisms underlying the pathogenesis of P. dispersa remain largely uninvestigated. In the present study, we describe a clinical infection caused by P. dispersa and provide the first genomic analysis of this bacterium.MethodsThe bacterial strain designated A003 was isolated from blood samples. Preliminary identification of strain A003 was conducted using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) technology (VITEK® MS, bioMérieux, France). Biochemical and antimicrobial susceptibility assessments were carried out utilizing the VITEK-2 compact automated microbial analysis system (bioMérieux, France). Additionally, whole genome sequencing and subsequent analysis were performed to further elucidate the potential pathogenicity.ResultsThe bacterial isolate was cultured overnight at 35°C in a CO2-enriched environment on Columbia blood agar, resulting in the appearance of a white, smooth, Gram-negative rod-shaped bacterium with diameters ranging from 1 to 2 mm. Identification of strain A003 was achieved with a high confidence level of 99.9% as P. dispersa using MALDI-TOF mass spectrometry. The average nucleotide identity (ANI) value between strain A003 and the reference strain P. dispersa DSM 30073T was 98.08%, while the DNA–DNA hybridization (DDH) value was 84.10% (Formula 2) based on genome sequencing data, which further confirmed that A003 belonged to the P. dispersa species. Comprehensive analysis revealed the presence of 372 virulence factors, 15 antibiotic resistance genes, 222 carbohydrate-active enzymes, and 666 genes related to Type III secretion system (T3SS) effector proteins, as identified through the core databases of VFDB (Virulence Factors of Pathogenic Bacteria), CARD (Comprehensive Antibiotic Resistance Database), CAZY (Carbohydrate-Active enZYmes Database), and T3SS. The virulence factors included type IV pili (TFP), type VI secretion system, flagella, iron uptake system, and ompA, which are implicated in bacterial pathogenicity. The antibiotic resistance profile indicated resistance to fluoroquinolones, cephalosporins, penams, macrolides, and aminoglycosides, as annotated by the CARD database.ConclusionThe draft genome sequenced represents the inaugural genomic sequence of P. dispersa derived from clinical sources. This advancement may enhance clinical practitioners’ comprehension of the organism’s clinical attributes and serve as a foundational resource for future research into its virulence, antibiotic resistance, and host–pathogen interactions.

Environmental alkalization suppresses deployment of virulence strategies in Pseudomonas syringae pv. tomato DC3000.

Plant pathogenic bacteria encounter a drastic increase in apoplastic pH during the early stages of plant immunity. The effects of alkalization on pathogen-host interactions have not been comprehensively characterized. Here, we used a global transcriptomic approach to assess the impact of environmental alkalization on Pseudomonas syringae pv. tomato DC3000 in vitro. In addition to the Type 3 Secretion System, we found expression of genes encoding other virulence factors such as iron uptake and coronatine biosynthesis to be strongly affected by environmental alkalization. We also found that the activity of AlgU, an important regulator of virulence gene expression, was induced at pH 5.5 and suppressed at pH 7.8, which are pH levels that this pathogen would likely experience before and during pattern-triggered immunity, respectively. This pH-dependent control requires the presence of periplasmic proteases, AlgW and MucP, that function as part of the environmental sensing system that activates AlgU in specific conditions. This is the first example of pH-dependency of AlgU activity, suggesting a regulatory pathway model where pH affects the proteolysis-dependent activation of AlgU. These results contribute to deeper understanding of the role apoplastic pH has on host-pathogen interactions.IMPORTANCEPlant pathogenic bacteria, like Pseudomonas syringae, encounter many environmental changes including oxidative stress and alkalization during plant immunity, but the ecological effects of the individual responses are not well understood. In this study, we found that transcription of many previously characterized virulence factors in P. syringae pv. tomato DC3000 is downregulated by the level of environmental alkalization these bacteria encounter during the early stages of plant immune activation. We also report for the first time the sigma factor AlgU is post-translationally activated by low environmental pH through its natural activation pathway, which partially accounts for the expression Type 3 Secretion System virulence genes at acidic pH. The results of this study demonstrate the importance of extracellular pH on global regulation of virulence-related gene transcription in plant pathogenic bacteria.

Constitutive activation of two-component systems reveals regulatory network interactions in Streptococcus agalactiae

Bacterial two-component systems (TCSs) are signaling modules that control physiology, adaptation, and host interactions. A typical TCS consists of a histidine kinase (HK) that activates a response regulator via phosphorylation in response to environmental signals. Here, we systematically test the effect of inactivating the conserved phosphatase activity of HKs to activate TCS signaling pathways. Transcriptome analyses of 14 HK mutants in Streptococcus agalactiae, the leading cause of neonatal meningitis, validate the conserved HK phosphatase mechanism and its role in the inhibition of TCS activity in vivo. Constitutive TCS activation, independent of environmental signals, enables high-resolution mapping of the regulons for several TCSs (e.g., SaeRS, BceRS, VncRS, DltRS, HK11030, HK02290) and reveals the functional diversity of TCS signaling pathways, ranging from highly specialized to interconnected global regulatory networks. Targeted analysis shows that the SaeRS-regulated PbsP adhesin acts as a signaling molecule to activate CovRS signaling, thereby linking the major regulators of host-pathogen interactions. Furthermore, constitutive BceRS activation reveals drug-independent activity, suggesting a role in cell envelope homeostasis beyond antimicrobial resistance. This study highlights the versatility of constitutive TCS activation, via phosphatase-deficient HKs, to uncover regulatory networks and biological processes.

Lumpy skin disease: a systematic review of mode of transmission, risk of emergence, and risk entry pathways.

Lumpy skin disease (LSD), a viral disease of cattle, can be acute, subacute, or inactive. It is distinguished by fever and the abrupt emergence of firm, confined cutaneous nodules that usually necrotize. Similar lesions may occur in the skeletal muscles and the mucosae of the digestive and respiratory tracts. It is an enzootic, rapidly explorative, and sometimes fatal infection, characterized by multiple raised nodules on the skin of infected animals. LSDV has a large genome, it is employed as a vaccine carrier, generating a new complex with other viral genes by homologous recombination. This review summarizes our current knowledge of lumpy skin disease (LSD), its impact on animal health, host-pathogen interaction, etiology, signs or symptoms, prevention, and treatment strategies.

Characterization of a High-Affinity Copper Transporter CTR1a in the White-Nose Syndrome Causing Fungal Pathogen Pseudogymnoascus destructans.

Copper is an essential micronutrient and the ability to scavenge tightly bound or trace levels of copper ions at the host-pathogen interface is vital for fungal proliferation in animal hosts. Recent studies suggest that trace metal ion acquisition is critical for the establishment and propagation of Pseudogymnoascus destructans, the fungal pathogen responsible for white-nose syndrome (WNS), on their bat host. However, little is known about these metal acquisition pathways in P. destructans. In this study, we report the characterization of the P. destructans high-affinity copper transporter VC83_00191 (PdCTR1a), which is implicated as a virulence factor associated with the WNS disease state. Using Saccharomyces cerevisiae as a recombinant expression host, we find that PdCTR1a can efficiently traffic Cu ions into the yeast cytoplasm. Complementary studies in the native P. destructans fungus provide evidence that PdCTR1a transcripts and protein levels are dictated by Cu-bioavailability in the growth media. Our study demonstrates that PdCTR1a is a functional high-affinity copper transporter and is relevant to Cu homeostasis pathways in P. destructans.

A human model of Buruli ulcer: Provisional protocol for a Mycobacterium ulcerans controlled human infection study.

Critical knowledge gaps have impeded progress towards reducing the global burden of disease due to Mycobacterium ulcerans, the cause of the neglected tropical disease Buruli ulcer (BU). Development of a controlled human infection model of BU has been proposed as an experimental platform to explore host-pathogen interactions and evaluate tools for prevention, diagnosis, and treatment. We have previously introduced the use case for a new human model and identified M. ulcerans JKD8049 as a suitable challenge strain. Here, we present a provisional protocol for an initial study, for transparent peer review during the earliest stages of protocol development. Following simultaneous scientific peer review and community/stakeholder consultation of this provisional protocol, we aim to present a refined protocol for institutional review board (IRB) evaluation.

Computational identification and characterization of chitinase 1 and chitinase 2 from neotropical isolates of Beauveria bassiana

BackgroundThe fungus Beauveria bassiana is widely used for agronomical applications, mainly in biological control. B. bassiana uses chitinase enzymes to degrade chitin, a major chemical component found in insect exoskeletons and fungal cell walls. However, until recently, genomic information on neotropical isolates, as well as their metabolic and biotechnological potential, has been limited.MethodsEight complete B. bassiana genomes of Neotropical origin and three references were studied to identify chitinase genes and its corresponding proteins, which were curated and characterized using manual curation and computational tools. We conducted a computational study to highlight functional differences and similarities for chitinase proteins in these Neotropical isolates.ResultsEleven chitinase 1 genes were identified, categorized as chitinase 1.1 and chitinase 1.2. Five chitinase 2 genes were identified but presented a higher sequence conservation across all sequences. Interestingly, physicochemical parameters were more similar between chitinase 1.1 and chitinase 2 than between chitinase 1.1 and 1.2.ConclusionChitinases 1 and 2 demonstrated variations, especially within chitinase 1, which presented a potential paralog. These differences were observed in their physical parameters. Additionally, CHIT2 completely lacks a signal peptide. This implies that CHIT1 might be associated with infection processes, while CHIT2 could be involved in morphogenesis and cellular growth. Therefore, our work highlights the importance of computational studies on local isolates, providing valuable resources for further experimental validation. Intrinsic changes within local species can significantly impact our understanding of complex pathogen-host interactions and offer practical applications, such as biological control.

A comprehensive review of integrated management strategies for damping-off disease in chili

Damping-off disease in chili (Capsicum annum L.) cultivation is a significant global issue, severely affecting seeds, seedlings, and young plants, regardless of the location of cultivation, whether in greenhouses or open fields. Despite chili being a widely popular vegetable used in various cuisines globally, farmers face challenges in meeting the growing demand due to the extensive damage caused by this disease, ranging from 20 to 85%. The shelf life and quality of mature pods are also severely affected. Damping-off disease is mainly caused by soil-borne fungus from the Pythium species, with additional contributions from Phytophthora, Fusarium, and Rhizoctonia species. These pathogens’ adaptability to diverse environmental conditions and resistance to synthetic fungicides make controlling damping-off on a commercial scale challenging. However, integrated disease management has shown promising results as a remedial approach. In this review, we discuss the current state of chili diseases, the nature of the pathogens causing damping-off, the epidemiology of the disease, and various control mechanisms. In this review, we broadly discuss the current state of chili diseases, the nature of the pathogens causing damping-off, the epidemiology of the disease, and various control mechanisms. Furthermore, we highlight the importance and efficacy of integrated disease management techniques, along with future prospects in unexplored areas, such as host–pathogen interaction and sustainable disease control measures. The information in this review aims to assist chili growers in understanding the epidemiology and management of damping-off in chili cultivation.

Integrative genomic, virulence, and transcriptomic analysis of emergent Streptococcus dysgalactiae subspecies equisimilis (SDSE) emm type stG62647 isolates causing human infections.

This study integrated genomic sequencing, mouse virulence assays, and bacterial transcriptomic analysis to advance our understanding of the molecular mechanisms contributing to Streptococcus dysgalactiae subsp. equisimilis emm type stG62647 pathogenesis. We tested a large cohort of genetically closely related stG62647 isolates for virulence using an established mouse model of necrotizing myositis and discovered a broad spectrum of virulence phenotypes, with near-mortality rates ranging from 20% to 95%. This variation was unexpected, given their close genetic proximity. Transcriptome analysis of stG62647 isolates responsible for the lowest and highest near-mortality rates suggested that these isolates used multiple molecular pathways to alter their virulence. In addition, some genes encoding transcriptional regulators and putative virulence factors likely contribute to SDSE emm type stG62647 pathogenesis. These data underscore the complexity of pathogen-host interactions in an emerging SDSE clonal group.

The role of the AHR in host-pathogen interactions.

Host-microorganism encounters take place in many different ways and with different types of outcomes. Three major types of microorganisms need to be distinguished: (1) pathogens that cause harm to the host and must be controlled; (2) environmental microorganisms that can be ignored but must be controlled at higher abundance; and (3) symbiotic microbiota that require support by the host. Recent evidence indicates that the aryl hydrocarbon receptor (AHR) senses and initiates signalling and gene expression in response to a plethora of microorganisms and infectious conditions. It was originally identified as a receptor that binds xenobiotics. However, it was subsequently found to have a critical role in numerous biological processes, including immunity and inflammation and was recently classified as a pattern recognition receptor. Here we review the role of the AHR in host-pathogen interactions, focusing on AHR sensing of different microbial classes, the ligands involved, responses elicited and disease outcomes. Moreover, we explore the therapeutic potential of targeting the AHR in the context of infection.

Animal Models in COVID-19 Research: Insights and Outcomes

Animal models have been critical in understanding the pathogenesis, transmission, and therapeutic interventions for COVID-19, caused by SARS-CoV-2. The primary challenges include species-specific differences in viral receptor expression, particularly the ACE2 receptor, and immune system responses, which can affect how closely these models mimic human COVID-19 pathology. Additionally, ethical concerns around the use of animals, especially non-human primates, and logistical issues such as high costs, housing requirements, and limited availability of certain models, further complicate research efforts. Transgenic mice expressing human ACE2 (hACE2) are widely used because of their ease of handling and cost-effectiveness. Other animal models include non-human primates, hamsters, cats, and ferrets, which were used to study viral transmission and host-pathogen interactions. The rapid dynamics of the SARSCoV-2 evolution is one of the challenges and the complementary use of multiple models provides a more comprehensive understanding of the disease. Nonetheless, these models have been essential for rapid advancements in COVID19 vaccine and therapeutic development, offering insights that continue to guide public health strategies. The review discusses the various animal models used in SARS-CoV-2 research, the outcomes, and the challenges and questions to be addressed Keywords: Animal models, SARS-CoV-2, COVID-19

SIV infection in sooty mangabeys does not impact survival but changes the relative frequency of the main cause of death.

In this study, we demonstrate, for the first time, that the natural, non-pathogenic SIV infection of the African monkey SM has a clinical impact which is revealed in terms of main causes of mortality, which are significantly different in the infected animals as compared to the uninfected ones. Indeed, SIV-infected SMs are at higher risk of dying of infectious diseases but appear to be somewhat protected from cardiovascular causes of death. The identification of a specific pattern of mortality associated with the infection suggests that the host-pathogen interaction between SIV and the SM immune system, while non-pathogenic in nature, has a detectable impact on the overall health status of the animals.

Alpha-hemolysin promotes internalization of Staphylococcus aureus into human lung epithelial cells via caveolin-1- and cholesterol-rich lipid rafts

Staphylococcus aureus is a pathogen associated with severe respiratory infections. The ability of S. aureus to internalize into lung epithelial cells complicates the treatment of respiratory infections caused by this bacterium. In the intracellular environment, S. aureus can avoid elimination by the immune system and the action of circulating antibiotics. Consequently, interfering with S. aureus internalization may represent a promising adjunctive therapeutic strategy to enhance the efficacy of conventional treatments. Here, we investigated the host-pathogen molecular interactions involved in S. aureus internalization into human lung epithelial cells. Lipid raft-mediated endocytosis was identified as the main entry mechanism. Thus, bacterial internalization was significantly reduced after the disruption of lipid rafts with methyl-β-cyclodextrin. Confocal microscopy confirmed the colocalization of S. aureus with lipid raft markers such as ganglioside GM1 and caveolin-1. Adhesion of S. aureus to α5β1 integrin on lung epithelial cells via fibronectin-binding proteins (FnBPs) was a prerequisite for bacterial internalization. A mutant S. aureus strain deficient in the expression of alpha-hemolysin (Hla) was significantly impaired in its capacity to enter lung epithelial cells despite retaining its capacity to adhere. This suggests a direct involvement of Hla in the bacterial internalization process. Among the receptors for Hla located in lipid rafts, caveolin-1 was essential for S. aureus internalization, whereas ADAM10 was dispensable for this process. In conclusion, this study supports a significant role of lipid rafts in S. aureus internalization into human lung epithelial cells and highlights the interaction between bacterial Hla and host caveolin-1 as crucial for the internalization process.

MSphere of Influence: Revisiting the central dogma, again!

Dr. Parimal Samir works in the field of host-pathogen interactions. In this mSphere of Influence article, he reflects on how the manuscript entitled "De novo gene synthesis by an antiviral reverse transcriptase" by Samuel Sternberg and colleagues made an impact by reminding him that there is still so much to discover in life sciences.

Molecular Dynamics Reveal Key Steps in BAR-Related Membrane Remodeling.

Endocytosis plays a complex role in pathogen-host interactions. It serves as a pathway for pathogens to enter the host cell and acts as a part of the immune defense mechanism. Endocytosis involves the formation of lipid membrane vesicles and the reshaping of the cell membrane, a task predominantly managed by proteins containing BAR (Bin1/Amphiphysin/yeast RVS167) domains. Insights into how BAR domains can remodel and reshape cell membranes provide crucial information on infections and can aid the development of treatment. Aiming at deciphering the roles of the BAR dimers in lipid membrane bending and remodeling, we conducted extensive all-atom molecular dynamics simulations and discovered that the presence of helix kinks divides the BAR monomer into two segments-the "arm segment" and the "core segment"-which exhibit distinct movement patterns. Contrary to the prior hypothesis of BAR domains working as a rigid scaffold, we found that it functions in an "Arms-Hands" mode. These findings enhance the understanding of endocytosis, potentially advancing research on pathogen-host interactions and aiding in the identification of new treatment strategies targeting BAR domains.

Chlamydia trachomatis Inc Ct226 is vital for FLI1 and LRRF1 recruitment to the chlamydial inclusion.

The obligate intracellular pathogen, Chlamydia trachomatis, establishes an intracellular niche within a host membrane-derived vacuole called the chlamydial inclusion. From within this inclusion, C. trachomatis orchestrates numerous host-pathogen interactions, in part, by utilizing a family of type III secreted effectors, termed inclusion membrane proteins (Incs). Incs are embedded within the inclusion membrane, and some function to recruit host proteins to the inclusion. Two such recruited host proteins are leucine rich repeat Flightless-1 interacting protein 1 (LRRF1/LRRFIP1) and its binding partner Flightless 1 (FLI1/FLII). Previously, LRRF1 has been shown to interact with Inc protein Ct226/CTL0478. This is the first study to examine interactions of FLI1 with candidate Incs or with LRRF1 during infection. We hypothesized that FLI1 recruitment to the inclusion would be dependent on LRRF1 localization. We demonstrated that FLI1 co-immunoprecipitated with Ct226 but only in the presence of LRRF1. Furthermore, FLI1 localized to the inclusion when LRRF1 was depleted via small interfering RNA, suggesting that FLI1 may have an alternative recruitment mechanism. We further developed a series of CRISPRi knockdown and complementation strains in C. trachomatis serovar L2 targeting ct226 and co-transcribed candidate Incs, ct225 and ct224. Simultaneous knockdown of ct226, ct225, and ct224 prevented localization of both FLI1 and LRRF1 to the inclusion, and only complementation of ct226 restored their localization. Thus, we demonstrated Ct226 is critical for FLI1 and LRRF1 localization to the inclusion. Our results also indicate an LRRF1-independent localization mechanism for FLI1, which likely influence their mechanism(s) of action during chlamydial infection.IMPORTANCEChlamydia trachomatis is a leading cause of both bacterial sexually transmitted infections and preventable infectious blindness worldwide. As an obligate intracellular pathogen, C. trachomatis has evolved multiple ways of manipulating the host to establish a successful infection. As such, it is important to understand host-chlamydial protein-protein interactions as these reveal strategies that C. trachomatis uses to shape its intracellular environment. This study looks in detail at interactions of two host proteins, FLI1 and LRRF1, during chlamydial infection. Importantly, the series of CRISPR inference knockdown and complement strains developed in this study suggest these proteins have both independent and overlapping mechanisms for localization, which ultimately will dictate how these proteins function during chlamydial infection.