Intracellular Bacteria Research Articles

Differential Transcriptomic Profile of Piscirickettsia salmonis LF-89 and EM-90 During an In Vivo Spatial Separation Co-Culture in Atlantic Salmon

Salmonid rickettsial septicemia (SRS) is a critical sanitary problem in the Chilean aquaculture industry since it induces the highest mortality rate in salmonids among all infectious diseases. Piscirickettsia salmonis, a facultative intracellular bacterium, is the biological agent of SRS. In Chile, two genogroups of P. salmonis, designated as LF-89 and EM-90, have been identified. Previous studies suggested that their cohabitation triggers the expression of virulence effectors, which may be related to a higher pathogenicity in salmonids during co-infection with both P. salmonis genogroups. Therefore, we aimed to evaluate if the physical contact between two isolates from LF-89 and EM-90 is necessary to activate this effect. Through a spatially separated in vivo co-culture inside Atlantic salmon (Salmo salar) post smolts and RNA-seq analysis, we compared the differentially expressed genes (DEGs) with previous results from an in vivo mixed co-culture. The results showed that although the LF-89-like isolate and the EM-90-like isolate had a similar DEG profile under both co-culture conditions, important virulence factors observed during the mixed co-cultures (i.e., flagellar-related genes, CydD, and NCS2) were absent in the spatially separated co-cultures. Hence, the synergistic effect linked to increased pathogenicity to the host may be driven by the physical co-localization and contact between the P. salmonis LF-89-like and EM-90-like isolates.

“Nano knife” for efficient piezocatalytic inactivation of amoeba spores and their intracellular bacteria: Synergetic effect between physical damage and chemical oxidation

Microbial interactions between infectious agents severely interfere with the disinfection process, and current disinfection methods are unable to effectively inactivate intracellular pathogens, posing a new threat to drinking water safety. In this study, we first reported the high efficiency of piezocatalysis in inactivating amoebae and their intracellular bacteria. Results showed that the inactivation rates of the MoS2/rGO piezocatalytic system for amoebic spores and their intracellular bacteria were 4.18 and 5.02-log, respectively, within 180min. Based on scavenger studies and ESR tests, the efficient inactivation of pathogens can be attributed to the generation of reactive oxygen species (ROS), and different pathogens exhibit varying tolerances to distinct ROS. Moreover, TEM analysis revealed that the sharp edge of MoS2/rGO was conducive to the physical cutting of amoeba's cell wall and membrane, promoting the attack of ROS and ensuring a more thorough deactivation. Additionally, the intracellular ROS produced by amoebae is not only conducive to the inactivation of amoebae but also the main reason for the inactivation of bacteria in spores. This study provides a new solution for the inactivation of amoeba spores and their intracellular bacteria and emphasizes the high efficiency of the synergistic effect of physical damage and chemical oxidation.

Campylobacterjejuni-derived cytolethal distending toxin promotes colorectal cancer metastasis.

Various forms of solid tumors harbor intracellular bacteria, but the physiological consequences of these microorganisms are poorly understood. We show that Campylobacter is significantly enriched in primary colorectal cancer (CRC) lesions from patients with metastasis. Campylobacterjejuni-derived cytolethal distending toxin (CDT) promotes CRC metastasis through JAK2-STAT3-MMP9 signaling in liver or pulmonary metastatic mice models, as confirmed in C.jejuni-infected human colonic tissue and CDT-treated colonic tumoroids from patients. Genetic deletion of cdtB (ΔcdtB) or purified CdtB protein demonstrates that the genotoxin is essential for C.jejuni's pro-metastatic property. In C.-jejuni-colonized mice, increased translocation of CDT-producing C.jejuni to extraintestinal implanted tumors potentially leads to accelerated metastasis of these tumors. Overall, these findings demonstrate that an intratumor-bacteria-derived genotoxin accelerates tumor metastasis, potentially opening a new diagnostic and therapeutic avenue for cancer management.

Omadacycline in the Treatment of Chlamydia psittaci Pneumonia: A Retrospective Study on Efficacy and Safety

Aims/Background Chlamydia psittaci is an obligate intracellular bacterium that primarily infects birds, but can cause respiratory infections in humans. Clinical evidence supporting the use of omadacycline for the treatment of Chlamydia psittaci pneumonia remains limited; therefore, this study aimed to evaluate the potential of omadacycline in treating Chlamydia psittaci pneumonia by analyzing the patients’ clinical outcomes and the drug safety profile. Methods We retrospectively reviewed the medical records of 15 patients with Chlamydia psittaci pneumonia treated at the First Affiliated Hospital, Zhejiang University School of Medicine between January and December 2023. Following diagnosis with the aid of metagenomic next-generation sequencing, the patients received omadacycline for treatment, and their clinical outcomes and laboratory marker profiles were monitored to assess the treatment efficacy and safety. Results Significant improvements were observed in clinical symptoms and laboratory markers, including C-reactive protein (p < 0.001), procalcitonin (p = 0.001), neutrophil percentage (p < 0.001), and the SpO2/FiO2 ratio (p < 0.001), after treatment with omadacycline. A 100% cure rate was reported within 28 days of treatment initiation, with gastrointestinal disturbances being the most common side effect. Conclusion Omadacycline shows promise in treating Chlamydia psittaci pneumonia and is well tolerated by the users. However, further controlled trials involving larger samples are required to confirm the efficacy and safety of the drug.

An exacerbated phosphate starvation response triggers Mycobacterium tuberculosis glycerol utilization at acidic pH.

The mechanisms controlling Mycobacterium tuberculosis (Mtb) replication and survival inside its human host remain ill-defined. Phagosome acidification and nutrient deprivation are common mechanisms used by macrophages to restrict the replication of intracellular bacteria. Mtb stops replicating at mildly acidic pH (<pH5.8), an adaptive process called "acid growth arrest," which is thought to play an important role in Mtb virulence. Using a genome-wide mutagenesis screen, we identified 95 genes whose disruption either decreases or increases Mtb fitness during acid growth arrest. We show that the virulence-associated inorganic phosphate (Pi) uptake system (Pst-1) regulates the ability of Mtb to replicate in acidic conditions. Deletion of pstA1, a gene coding for a subunit of the Pst-1 system, results in the overexpression of the Pi starvation regulator regX3, which is sufficient to restore Mtb growth in acid conditions. Our data further support the role of limited glycerol uptake and ROS-mediated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition in causing Mtb acid growth arrest. This study reveals an unexpected role of the Pi starvation response in regulating acid growth arrest and highlights the intricacy of the mechanisms regulating redox homeostasis, acid stress response, and nutrient utilization in Mtb.IMPORTANCEDespite the availability of antibiotic treatment, M. tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains a major infectious disease killer worldwide. A better understanding of the environments that Mtb faces during infection and the mechanisms Mtb employs to respond and adapt may help identify currently unexplored pathways and targets for the development of novel anti-TB drugs. Here, we demonstrate that Mtb growth in acid can be restored by the over-expression of the Pi starvation response regulator regX3. This work paves the way toward a better understanding of the mechanisms controlling Mtb growth at acidic pH and highlights the role of inorganic phosphate in this process.

Differential vasoproliferative traits of Bartonella henselae strains associated with autotransporter BafA variants.

Bartonella henselae, a Gram-negative facultative intracellular bacterium, is the etiological agent of cat-scratch disease and also causes bacillary angiomatosis in immunocompromised individuals. Although the ability to promote vascular endothelial cell proliferation differs among Bartonella species, variations among strains within B. henselae remain unclear. Bartonella angiogenic factor A (BafA) and Bartonella adhesin A (BadA) have been identified as autotransporters of B. henselae that are involved in endothelial cell proliferation. Although strain-specific differences in the expression of BadA and the VirB/D4 type IV secretion system have been reported, BafA expression among B. henselae strains has yet to be examined. Therefore, the present study investigated the proliferation-promoting ability of 13 B. henselae strains from several sources in human umbilical vein endothelial cells (HUVECs). We identified BafA variants 1 and 2 based on the deduced amino acid sequences of its passenger domain. The recombinant proteins of both variants exhibited similar proliferation activity against HUVECs. However, BafA variant 2 strains showed cytotoxicity at a high bacterial inoculum in a direct coculture with HUVECs, which was attenuated in an indirect coculture. These strains, in contrast to BafA variant 1 strains, highly expressed BadA and exhibited bacterial aggregation. Based on a core genome SNP analysis of 50 B. henselae strains, the BafA variant types corresponded to clades 1-4. These results indicate that vasoproliferative traits differ among B. henselae clades based on the variant types. Therefore, this study provides a new conceptual framework in which the clades of B. henselae may predict their pathogenicity in humans.IMPORTANCEBartonella species including Bartonella henselae, Bartonella quintana, and Bartonella bacilliformis cause vasoproliferative lesions. Their proliferation-promoting ability in vascular endothelial cells differs among Bartonella species; however, it is unclear whether these differences exist among B. henselae strains. We herein showed that B. henselae strains exhibited variable proliferation-promoting ability and cytotoxicity in vascular endothelial cells, which corresponded to the bafA gene variants possessed by the strains. The expression levels of Bartonella angiogenic factor A (BafA) and Bartonella adhesin A, as well as the degree of proliferation-promoting ability and cytotoxicity in endothelial cells, varied among the strains. A core genome SNP analysis of strains using whole genome sequencing data divided B. henselae strains into four clades, with each clade corresponding to BafA variants 1-4. These results suggest the differential vasoproliferative potency of B. henselae, with potential implications in clinical management, including risk stratification and predictions of the clinical course.

RNF144A inhibits autophagy by targeting BECN1 for degradation during L. monocytogenes infection

ABSTRACT Listeria monocytogenes (L. monocytogenes, Lm) is widely used in the laboratory as an infection model for the research on pathogenesis and host defense against gram-positive intracellular bacteria. Macroautophagy (called simply “autophagy” hereafter), is important in the host defense against pathogens, such as bacteria, viruses, and parasites. BECN1 plays a pivotal role in the initiation of autophagy and accumulating evidence indicates that post-translational modifications of BECN1 provide multiple strategies for autophagy regulation. In this study, we demonstrated that the RING1-IBR-RING2 (RBR) family member RNF144A (ring finger protein 144A), which was induced by Lm infection, promoted Lm infection in an autophagy-dependent but STING1-independent pattern. rnf144a deficiency in mice protected mice from Lm infection with inhibited innate immune responses. Interestingly, RNF144A decreased Lm-induced autophagosome accumulation. Mechanistic investigation indicated that RNF144A interacted with BECN1 and promoted its K48-linked ubiquitination, leading to the subsequent proteasome-dependent degradation of BECN1 and reduced autophagosome accumulation. Further study demonstrated that RNF144A promoted the ubiquitination of BECN1 at K117 and K427, and these two ubiquitination sites were essential to the role of BECN1 in autophagy and Lm infection. Thus, our findings suggested a new regulator in intracellular bacterial infection and autophagy, which may contribute to our understanding of host defense against intracellular bacterial infection via autophagy. Abbreviations: ATG3: autophagy related 3; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG10: autophagy related 10; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; Baf A1: bafilomycin A1; BECN1: beclin 1; BMDC: bone marrow-derived dendritic cell; BMDM: bone marrow-derived macrophage; CFUs: colony-forming units; CHX: cycloheximide; CQ: chloroquine; CXCL10/IP-10: C-X-C motif chemokine ligand 10; EBSS: Earle’s balanced salt solution; ELISA: enzyme-linked immunosorbent assay; IFIT1/ISG56: interferon induced protein with tetratricopeptide repeats 1; IFNB/IFN-β: interferon beta; IL6: interleukin 6; IRF3, interferon regulatory factor 3; Lm: L. monocytogenes; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MOI: multiplicity of infection; PLA: proximity ligation assay; PMA: phorbol myristate acetate; PMA-THP1, PMA-differentiated THP1; PMs: peritoneal macrophages; PTMs: posttranslational modifications; RBR: RING1-IBR-RING2; RNF144A: ring finger protein 144A; STING1, stimulator of interferon response cGAMP interactor 1; TBK1, TANK binding kinase 1; TNF/TNF-α: tumor necrosis factor;

Detection of Rickettsia amblyommatis in Amblyomma mixtum and Rhipicephalus microplus Ticks Collected from Cattle in Tamaulipas, Mexico.

Background: Rickettsia spp. are obligate intracellular bacteria of utmost importance for public health and the economy since their presence can generate significant losses in livestock production, affecting the health of animals, the quality of the meat, milk, and other products derived from affected animals. Therefore, prevention of the transmission of these pathogens, their early identification and timely treatment are essential to mitigate their impact on public health and animal production. Materials and Methods: In the present work, hard ticks were collected from infested Bos indicus cattle in Aldama, Tamaulipas, Mexico. They were identified by morphology using dichotomous keys and by sequencing and analyzing of a fragment of the mitochondrial 16S. PCR was performed using specific primers targeting Rickettsia sp. gltA. Phylogenetic analyses were performed, aligning the amino acid sequences with Muscle, and a phylogenetic tree was generated using PhyML. Results and Conclusions: Amblyomma mixtum and Rhipicephalus microplus ticks infesting the cattle were identified. Using molecular techniques, the presence of Rickettsia amblyommatis was identified in the cohort of analyzed ticks, suggesting a circulation of this pathogen in livestock in this region and granting further research in this area.

Pangenome-Wide Association Study in the Chlamydiaceae Family Reveals Key Evolutionary Aspects of Their Relationship with Their Hosts

The Chlamydiaceae are a family of obligate intracellular bacteria known for their unique biphasic developmental cycle. Chlamydial are associated with various host organisms, including humans, and have been proposed as emerging pathogens. Genomic studies have significantly enhanced our understanding of chlamydial biology, host adaptation, and evolutionary processes. In this study, we conducted a complete pangenome association analysis (pan-GWAS) using 101 genomes from the Chlamydiaceae family to identify differentially represented genes in Chlamydia and Chlamydophila, revealing their distinct evolutionary strategies for interacting with eukaryotic hosts. Our analysis identified 289 genes with differential abundance between the two clades: 129 showed a strong association with Chlamydia and 160 with Chlamydophila. Most genes in Chlamydia were related to the type III secretion system, while Chlamydophila genes corresponded to various functional categories, including translation, replication, transport, and metabolism. These findings suggest that Chlamydia has developed a high dependence on mammalian cells for replication, facilitated by a complex T3SS for intracellular manipulation. In contrast, the metabolic and functional diversity in Chlamydophila allows it to colonize a broad range of hosts, such as birds, reptiles, amphibians, and mammals, making it a less specialized clade.

Overexpressing the ClpC AAA+ unfoldase accelerates developmental cycle progression in Chlamydia trachomatis.

Chlamydia is an obligate intracellular bacterium that undergoes a complex biphasic developmental cycle, alternating between the smaller, infectious, non-dividing elementary body (EB) and the larger, non-infectious but dividing reticulate body. Due to the differences between these functionally and morphologically distinct forms, we hypothesize protein degradation is essential to chlamydial differentiation. The bacterial Clp system, consisting of an ATPase unfoldase (e.g., ClpX or ClpC) and a proteolytic component (e.g., ClpP), is critical for the physiology of bacteria through its recognition, and usually degradation, of specific substrates. We observed by transmission electron microscopy that overexpression of wild-type ClpC, but not an ATPase mutant isoform, in Chlamydia increased glycogen accumulation within the vacuolar niche of the bacteria earlier in the developmental cycle than typically observed. This suggested ClpC activity may increase the expression of EB-associated genes. Consistent with this, targeted RT-qPCR analyses demonstrated a significant increase in several EB-associated gene transcripts earlier in development. These effects were not observed with overexpression of the ATPase mutant of ClpC, providing strong evidence that the activity of ClpC drives secondary differentiation. By analyzing the global transcriptional response to ClpC overexpression using RNA sequencing, we observed a shift to earlier expression of canonical late developmental cycle genes and other EB-associated genes. Finally, we directly linked overexpression of ClpC with earlier production of infectious chlamydiae. Conversely, disrupting normal ClpC function with an ATPase mutant caused a delay in developmental cycle progression. Overall, these findings provide the first mechanistic insight for initiation of secondary differentiation in Chlamydia.IMPORTANCEChlamydia species are obligate intracellular bacteria that require a host cell in which to complete their unique developmental cycle. Chlamydia differentiates between an infectious but non-replicating form, the elementary body, and a non-infectious but replicating form, the reticulate body. The signals that drive differentiation events are not characterized. We hypothesize that proteases are essential for mediating differentiation by allowing remodeling of the proteome as the organism transitions from one functional form to another. We previously reported that the Caseinolytic protease (Clp) system is essential for chlamydial growth. Here, we reveal a surprising function for ClpC, an unfoldase, in driving production of infectious chlamydiae during the chlamydial developmental cycle.

Molecular and Serological Findings in Sheep During Two Coxiella burnetii Outbreaks in Sicily (Southern Italy).

Q fever is a widespread zoonotic disease caused by the obligate intracellular bacterium Coxiella burnetii, primarily transmitted through the inhalation of contaminated aerosols. This study aimed to detect C. burnetii in two Sicilian sheep flocks, with no better defined reproductive disorders reported by the farmers. Blood, individual and bulk milk, ticks, and conjunctival swabs were collected from both flocks (A and B). Real-time and traditional PCRs were carried out to detect C. burnetii DNA and anti-C. burnetii antibodies were searched using an ELISA. In terms of Farm A, C. burnetii DNA was detected in 7.1% of blood samples, 20% of individual milk samples, bulk milk, 66.6% of conjunctival swabs, and in all the examined tick pools. Anti-C. burnetii antibodies were found in 77.0% of sera, 92.5% of individual milk samples, and bulk milk. In terms of Farm B, C. burnetii DNA was detected in 3.8% of blood samples, 39.4% of individual milk samples, bulk milk, 100% of conjunctival swabs, and in all tick pools; anti-C. burnetii antibodies were present in 53.6% of sera, 73.2% of milk samples, and in bulk milk. Our results highlight the high diffusion of C. burnetii in the two outbreaks, with widespread pathogen circulation, significant shedding in dairy products, and high environmental contamination, highlighting the need for enhanced surveillance and control measures in dairy sheep farms.

PH-Responsive Protein-Polycation Nanocarriers for Efficient Eradication of Bacterial Biofilms and Intracellular Bacteria.

Bacterial biofilms and intracellular pathogens pose significant challenges in eradication, often leading to persistent infections that are difficult to treat. To address this issue, the hydrophobic biofilm dispersant D-tyrosine is encapsulated within protein-polycation nanoparticles, designed using a mannose-terminated cationic polymer and concanavalin through electrostatic interactions. Thermodynamic studies reveal that free mannosyl groups on the nanoparticle surface promote spontaneous binding to receptor molecules mimicking those on bacterial biofilms and host cells. Under mildly acidic conditions, the nanoparticles reduce in size from 550 to ≈48 nm within 2 h, releasing 76% of encapsulated D-tyrosine. The combination of mannose targeting, particle size reduction, and controlled D-tyrosine release enable the nanoparticles to eliminate 70%-80% of the Pseudomonas aeruginosa and Staphylococcus aureus biofilm biomass at minimum bactericidal concentration (MBC) and 2MBC while eradicating 8 log of bacteria embedded within the biofilm. In an intracellular Pseudomonas aeruginosa infection model using RAW 264.7 macrophages, the nanoparticles at 2MBC eliminate over 95% of the intracellular bacteria without inducing an increase in the inflammatory cytokine interleukin-6. These protein-polycation nanoparticles, which activate their antimicrobial properties under acidic conditions, efficiently penetrate bacterial biofilms and host cell barriers via their mannose-rich surface, offering a promising strategy for the treatment of persistent infections.

Antibacterial Mechanisms and Clinical Impact of Sitafloxacin.

Sitafloxacin is a 4th generation fluoroquinolone antibiotic with broad activity against a wide range of Gram-negative and Gram-positive bacteria. It is approved in Japan and used to treat pneumonia and urinary tract infections (UTIs) as well as other upper and lower respiratory infections, genitourinary infections, oral infections and otitis media. Compared to other fluoroquinolones, sitafloxacin displays a low minimal inhibitory concentration (MIC) for many bacterial species but also activity against anaerobes, intracellular bacteria, and persisters. Furthermore, it has also shown strong activity against biofilms of P. aeruginosa and S. aureus in vitro, which was recently validated in vivo with murine models of S. aureus implant-associated bone infection. Although limited in scale at present, the published literature supports the further evaluation of sitafloxacin in implant-related infections and other biofilm-related infections. The aim of this review is to summarize the chemical-positioning-based mechanisms, activity, resistance profile, and future clinical potential of sitafloxacin.

Role of Oral Yeast in Replenishing Gastric Mucosa with Yeast and Helicobacter pylori.

The relationship between oral and gastric yeasts and their role in the colonization of Helicobacter pylori in the stomach was studied. Four groups of 221, 7, 44, and 10 patients were used for the isolation of H. pylori and oral and gastric yeasts. In Group 1, gastric biopsies were used for the isolation of H. pylori and yeast, rapid urease test (RUT), staining with Gram's and hematoxylin & eosin (H&E), and immunohistochemistry (IHC) methods. In the other three groups, DNAs extracted from H. pylori and yeasts were used for the amplification of H. pylori-specific genes. Wet mounts of yeasts in Group 2 were examined to observe intracellular bacteria and released EVs. Among 221 patients, 65 (29.3%) had oral yeast, 35 (15.8%) H. pylori, and 31 (14%) gastric yeast. Culture of oral yeasts showed a significant correlation with the detection of H. pylori by IHC (10.3%), Gram stain (9%), RUT (6.3%), H&E (4.9%), and culture (4%) (p < 0.05). Gram-stained biopsies showed the occurrence of yeast and H. pylori, and the release of EVs from yeast. Detection of similar H. pylori genes in oral and gastric yeasts from patients in Group 2 showed their common source. Oral yeasts in Groups 3 and 4 also carried H. pylori genes. Wet mount preparations of yeasts showed intracellular bacteria inside the yeast vacuole and the release of EVs that could carry H. pylori. Oral yeast protects its intracellular H. pylori and releases it inside EVs to safely reach gastric mucosa. Yeast, as the environmental reservoir of H. pylori, plays a crucial role in bacterial reinfection after successful eradication.

InSeq analysis of defined Legionella pneumophila libraries identifies a transporter-encoding gene cluster important for intracellular replication in mammalian hosts.

Legionella pneumophila is an intracellular bacterial pathogen that replicates inside human alveolar macrophages to cause a severe pneumonia known as Legionnaires' disease. L. pneumophila requires the Dot/Icm Type IV secretion system to deliver hundreds of bacterial proteins to the host cytosol that manipulate cellular processes to establish a protected compartment for bacterial replication known as the Legionella-containing vacuole. To better understand mechanisms apart from the Dot/Icm system that support survival and replication in this vacuole, we used transposon insertion sequencing in combination with defined mutant sublibraries to identify L. pneumophila fitness determinants in primary mouse macrophages and the mouse lung. This approach validated that many previously identified genes important for intracellular replication were critical for infection of a mammalian host. Further, the screens uncovered additional genes contributing to L. pneumophila replication in mammalian infection models. This included a cluster of seven genes in which insertion mutations resulted in L. pneumophila fitness defects in mammalian hosts. Generation of isogenic deletion mutants and genetic complementation studies verified the importance of genes within this locus for infection of mammalian cells. Genes in this cluster are predicted to encode nucleotide-modifying enzymes, a protein of unknown function, and an atypical ATP-binding cassette (ABC) transporter with significant homology to multidrug efflux pumps that has been named Lit, for Legionella infectivity transporter. Overall, these data provide a comprehensive overview of the bacterial processes that support L. pneumophila replication in a mammalian host and offer insight into the unique challenges posed by the intravacuolar environment.IMPORTANCEIntracellular bacteria employ diverse mechanisms to survive and replicate inside the inhospitable environment of host cells. Legionella pneumophila is an opportunistic human pathogen and a model system for studying intracellular host-pathogen interactions. Transposon sequencing is an invaluable tool for identifying bacterial genes contributing to infection, but current animal models for L. pneumophila are suboptimal for conventional screens using saturated mutant libraries. This study employed a series of defined transposon mutant libraries to identify determinants of L. pneumophila fitness in mammalian hosts, which include a newly identified bacterial transporter called Lit. Understanding the requirements for survival and replication inside host cells informs us about the environment bacteria encounter during infection and the mechanisms they employ to make this environment habitable. Such knowledge will be key to addressing future challenges in treating infections caused by intracellular bacteria.

Targeted Sonodynamic Therapy Platform for Holistic Integrative Helicobacter pylori Therapy.

Helicobacter pylori (H. pylori) is a primary pathogen associated with gastrointestinal diseases, including gastric cancer. The increase in resistance to antibiotics, along with the adverse effects caused by complicated medication protocols, has made the eradication of H. pylori a more formidable challenge, necessitating alternative therapeutics. Herein, a targeted nanoplatform is reported based on sonodynamic therapy, the chitosan-conjugated fucose loaded with indocyanine green (ICG@FCS). It penetrates the gastric mucosa and homes in on H. pylori through dual targeting mechanisms: molecular via fucose and physical via ultrasound. Upon ultrasound activation, it generates singlet oxygen, effectively attacking planktonic bacteria, disrupting biofilms, and facilitating the clearance of intracellular bacteria by promoting autophagy, including multidrug-resistant strains. The ICG@FCS nanoplatform minimally affects the gut microbiota and aids in gastric mucosa repair. a holistic integrative H. pylori therapy strategy is proposed that targets eradication while preserving gastrointestinal health. This strategy emphasizes the importance of maintaining patient health while eradicating the pathogen. This advancement is set to refine the comprehensive antibacterial approach, offering a promising horizon in the ongoing battle against antibiotic resistance and more effective gastric cancer prevention strategies.

Orientia tsutsugamushi infection reduces host gluconeogenic but not glycolytic substrates.

Orientia tsutsugamushi a causal agent of scrub typhus, is an obligate intracellular bacterium that, akin to other rickettsiae, is dependent on host cell-derived nutrients for survival and thus pathogenesis. Based on limited experimental evidence and genome-based in silico predictions, O. tsutsugamushi is hypothesized to parasitize host central carbon metabolism (CCM). Here, we (re-)evaluated O. tsutsugamushi dependency on host cell CCM as initiated by glucose and glutamine. Orientia infection had no effect on host glucose and glutamine consumption or lactate accumulation, indicating no change in overall flux through CCM. However, host cell mitochondrial activity and ATP levels were reduced during infection and correspond with lower intracellular glutamine and glutamate pools. To further probe the essentiality of host CCM in O. tsutsugamushi proliferation, we developed a minimal medium for host cell cultivation and paired it with chemical inhibitors to restrict the intermediates and processes related to glucose and glutamine metabolism. These conditions failed to negatively impact O. tsutsugamushi intracellular growth, suggesting the bacterium is adept at scavenging from host CCM. Accordingly, untargeted metabolomics was utilized to evaluate minor changes in host CCM metabolic intermediates across O. tsutsugamushi infection and revealed that pathogen proliferation corresponds with reductions in critical CCM building blocks, including amino acids and TCA cycle intermediates, as well as increases in lipid catabolism. This study directly correlates O. tsutsugamushi proliferation to alterations in host CCM and identifies metabolic intermediates that are likely critical for pathogen fitness.IMPORTANCEObligate intracellular bacterial pathogens have evolved strategies to reside and proliferate within the eukaryotic intracellular environment. At the crux of this parasitism is the balance between host and pathogen metabolic requirements. The physiological basis driving O. tsutsugamushi dependency on its mammalian host remains undefined. By evaluating alterations in host metabolism during O. tsutsugamushi proliferation, we discovered that bacterial growth is independent of the host's nutritional environment but appears dependent on host gluconeogenic substrates, including amino acids. Given that O. tsutsugamushi replication is essential for its virulence, this study provides experimental evidence for the first time in the post-genomic era of metabolic intermediates potentially parasitized by a scrub typhus agent.

Race for the surface between THP-1 macrophages and Staphylococcus aureus on various titanium implants with well-defined topography and wettability.

Gristina et al. (1987) suggested the fate of a biomaterial is decided in a “race for the surface” between pathogens and the host. To gain deeper insight into the mechanisms behind this concept, we investigated the “race for the surface” across three co-culture scenarios with THP-1 macrophages and Staphylococcus aureus (1:1 ratio), varying the order of addition: (i) simultaneous, (ii) macrophages first, and (iii) S. aureus first, on six Ti6Al4V-ELI surfaces modified with specific topographies and wettability. The outcome of the race for the surface was not influenced by these biomaterials but by the chronological introduction of macrophages and S. aureus. When macrophages and S. aureus arrived simultaneously, macrophages won the race, leading to the lowest number of viable S. aureus through rapid phagocytosis and killing. When macrophages arrived and established first, macrophages still prevailed but under greater challenge resulting from the lower bacterial killing efficiency of adherent macrophages and numerous viable intracellular bacteria, supporting the concept of the so-called immunocompromised zone around implants (upregulation of TLR-2 receptor and pro-inflammatory IL-1β). When S. aureus arrived first establishing a biofilm, bacteria won the race, leading to macrophage dysfunction and cell death (upregulation of FcγR and TLR-2 receptors, NF-κB signaling, NOX2 mediated reactive oxygen species), contributing to a persistent biofilm phenotype (upregulation of clfA, icaA, sarA, downregulation of agrA, hld, lukAB) and intracellular survival of S. aureus (lipA upregulation). The clinical implications are bacterial colonization of the implant and persistence of intracellular bacteria in periprosthetic tissues, which can lead to infection chronicity. Statement of SignificanceGristina et al. (1987) suggested the fate of a biomaterial is decided in a “race for the surface” between bacterial pathogens and host cells. There is a lack of in vitro co-culture models and knowledge on macrophage-S. aureus interactions on biomaterial surfaces, and none has evaluated the expression of virulence factors in S. aureus biofilms.We have successfully developed co-culture models and molecular panels and elucidated important cellular and molecular interactions between macrophages and S. aureus on a broad range of titanium biomaterials with well-defined surface topography and wettability. Our findings highlight the critical role of biofilm formation and the chronological order of bacteria or macrophage arrival in determining the fate of the surface.

Impact of transinfection of Wolbachia from the planthopper Laodelphax striatellus on reproductive fitness and transcriptome of the whitefly Bemisia tabaci

The whitefly Bemisia tabaci is critical global pest threatening crops and leading to agricultural losses. Wolbachia is an intracellular symbiotic bacterium in insects, which can regulate the growth and development of the host through various ways. In a prior study, Wolbachia was found to be transferred to whitefly and induce fitness changes. However, little is known about the underlying mechanisms of host-Wolbachia interactions in B. tabaci. In this study, a Wolbachia strain wStri was isolated from the small brown planthopper, Laodelphex striatellus, and transferred to B. tabaci. The distribution of Wolbachia in whiteflies was determined using fluorescence in situ hybridization. Reciprocal crossing experiments demonstrated that wStri did not induce cytoplasmic incompatibility phenotypes in B. tabaci, but prolonged the developmental duration of the offspring. We performed transcriptomic analysis of Wolbachia-infected female and male adults using Illumina-based RNA-Seq. A total of 843 differentially expressed genes (DEGs) were identified in infected females, among them 141 were significantly up-regulated and 702 were down-regulated by Wolbachia infection. In infected males, of 511 gene sets, 279 host genes were significantly up-regulated, and 232 were down-regulated by Wolbachia infection. KEGG analysis of DEGs demonstrated significant differences in gene pathway distribution between up-regulated and down-regulated genes. These genes are involved in various biological processes, including, but not limited to, detoxification, oxidation–reduction, metabolic processes, and immunity. The transcriptomic profiling of this study offers valuable information on the differential expression of genes in whiteflies following Wolbachia infection, and enhances our understanding of this host-symbiotic interaction.

Deciphering Rickettsia conorii metabolic pathways: a treasure map to therapeutic targets

Indian tick typhus is an infectious disease caused by intracellular gram-negative bacteria Rickettsia conorii (R. conorii). The bacterium is transmitted to humans through bite of infected ticks and sometimes by lice, fleas or mites. The disease is restricted to some areas with few cases but in last decade it is re-emerging with large number of cases from different areas of India. The insight in to genetic makeup of bacterial pathogens can be derived from their metabolic pathways. In the current study 18 metabolic pathways were found to be unique to the pathogen (R. conorii). A comprehensive analysis revealed 163 proteins implicated in 18 unique metabolic pathways of R. conorii. 140 proteins were reported to be essential for the bacterial survival, 46 were found virulent and 10 were found involved in resistance which can enhance the bacterial pathogenesis. The functional analysis of unique metabolic pathway proteins showed the abundance of plasmid conjugal transfer TrbL/VirB6, aliphatic acid kinase short chain, signal transduction response regulator receiver and components of type IV transporter system domains. The proteins were classified into six broad categories on the basis of predicted domains, i.e., metabolism, transport, gene expression and regulation, antimicrobial resistance, cell signalling and proteolysis. Further, in silico analysis showed that 88 proteins were suitable therapeutic targets which do not showed homology with host proteins. The 43 proteins showed hits with the DrugBank database showing their druggable nature and remaining 45 proteins were classified as novel drug targets that require further validation. The study will help to provide the better understanding of pathogens survival and embark on the development of successful therapies for the management of Indian tick typhus.