Intracellular Bacilli Research Articles

An arylsulfonamide that targets cell wall biosynthesis in Mycobacterium tuberculosis.

We investigated the mechanism of action of an arylsulfonamide with whole-cell activity against Mycobacterium tuberculosis. We newly synthesized the molecule and confirmed it had activity against both extracellular and intracellular bacilli. The molecule had some activity against HepG2 cells but maintained some selectivity. Bacterial cytological profiling suggested that the mechanism of action was via disruption of cell wall synthesis, with similarities to an inhibitor of the mycolic acid exporter MmpL3. The compound induced expression from the IniB promoter and caused a boost in ATP production but did not induce reactive oxygen species. A mutation in MmpL3 (S591I) led to low-level resistance. Taken together, these data confirm the molecule targets cell wall biosynthesis with MmpL3 as the most probable target.

Robust anti-tubercular profile of Solanum virginianum extract in enhancing isoniazid bioavailability and curtailing stress tolerance in Mycobacterium smegmatis.

The formidable survival mechanisms employed by Mycobacterium tuberculosis (Mtb), combined with the low bioavailability of anti-tubercular drugs and their associated hepatotoxicity, worsen tuberculosis management. Traditional medicinal plants offer potential solutions to these challenges. This study focuses on exploring the anti-tubercular potential of Solanum virginianum against Mycobacterium smegmatis, mc2155. HPTLC and UHPLC phytochemically characterized the hydro-methanolic extract of Solanum virginianum (SVE). SVE curtails the growth and viability of mc2155 under normal and in vitro stress conditions. The compromised cell wall integrity of mc2155 with SVE is depicted through scanning electron microscopy (SEM) while EtBr permeability assays and TLC-based comparative changes in lipids extraction addressed the integrity of the cell wall. Furthermore, SVE augmented the susceptibility of mc2155 towards Isoniazid (INH) through enhanced bioavailability. Adjunct treatment of SVE with INH demonstrated a markedly reduced survival of the intracellular bacilli. The study also uncovered the hepatoprotective potential of SVE in HepG2 cells. This research paves the way for deeper exploration into the potential of Solanum virginianum against virulent Mtb strains, emphasizing over the significance of traditional medicinal plants in tuberculosis treatment. Collectively, the findings suggest SVE as a potent candidate for independent or adjunct anti-tubercular therapy.

First report of Bartonella henselae and Bartonella clarridgeiae carriage in stray cats from Ecuador and its link to a cat scratch disease outbreak in 2022

IntroductionThe genus Bartonella includes species and subspecies of fastidious, facultative intracellular Gram-negative bacilli that infect a wide variety of mammalian reservoirs including cats and humans. In 2022, the Ecuadorian Ministry of Health reported an outbreak of cat scratch disease caused by B. henselae in the city of Guayaquil. Therefore, we aimed to characterize the presence of Bartonella spp. in domestic and stray cats from the area of Guayaquil where the outbreak happened in 2022. MethodsWhole blood samples of 100 domestic and stray cats were collected. Riboflavin synthase (ribC) and 16S rRNA genes detection was performed by PCR using Bartonella spp. specific primers, followed by Sanger sequencing and phylogenetic analysis. Results14 cats were positive for Bartonella spp. carriage. Phylogenetic analysis confirmed the presence of 12 cats infected with B. henselae and 2 cats with B. clarridgeiae. ConclusionsThere is a high prevalence of Bartonella spp. carriage in cats in the city of Guayaquil within the area where a recent cat scratch disease outbreak happened. Considering the high presence of cats and other domestic and stray animals in the city of Guayaquil, a One Health approach for surveillance and prevention of zoonotic diseases like cat scratch disease is needed.

Liposome-siderophore conjugates loaded with moxifloxacin serve as a model for drug delivery against Mycobacterium tuberculosis

Tuberculosis (TB) is an infectious disease that annually affects millions of people, and resistance to available antibiotics has exacerbated this situation. Another notable characteristic of Mycobacterium tuberculosis, the primary causative agent of TB, is its ability to survive inside macrophages, a key component of the immune system. In our quest for an effective and safe treatment that facilitates the targeted delivery of antibiotics to the site of infection, we have proposed a nanotechnology approach based on an iron chelator. Iron chelators are the primary mechanism by which bacteria acquire iron, a metal essential for their metabolism. Four liposomes were synthesized and characterized using the dynamic light scattering technique (DLS), nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). All of these methods revealed the presence of spherical particles, approximately 200 nm in size. NTA indicated a concentration of around 1011 particles/mL. We also developed and validated a high-performance liquid chromatography method for quantifying Moxifloxacin to determine encapsulation efficiency (EE) and release profiles (RF). The EE was 51.31 % for LipMox and 45.76 % for LipIchMox. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) confirmed the phagocytosis of liposomal vesicles by macrophages. Functionalizing liposomes with iron chelators can offer significant benefits for TB treatment, such as targeted drug delivery to intracellular bacilli through the phagocytosis of liposomal particles by cells like macrophages.

Human mesenchymal stromal cells inhibit Mycobacterium avium replication in clinically relevant models of lung infection

IntroductionNovel therapeutic strategies are urgently needed for Mycobacterium avium complex pulmonary disease (MAC-PD). Human mesenchymal stromal cells (MSCs) can directly inhibit MAC growth, but their effect on intracellular bacilli is...

Interleukin-26 expression in tuberculosis disease and its regulatory effect in macrophage polarization and intracellular elimination of Mycobacterium tuberculosis.

Tuberculosis(TB), an infectious disease caused by Mycobacterium tuberculosis (Mtb) infections, remains the leading cause of mortality from a single infectious agent globally. The progression of tuberculosis disease is contingent upon the complex interplay between the host's immune system and the pathogen Mtb. Interleukin-26 (IL-26), the most recently identified cytokine belonging to the IL-10 family, exhibits both extracellular antimicrobial properties and pro-inflammatory functions. However, the precise role of IL-26 in the host immune defense against Mtb infections and intracellular killing remains largely unexplored. In this study, we observed significantly elevated IL-26 mRNA expression in peripheral blood mononuclear cells of active-TB patients compared to healthy individuals. Conversely, circulating IL-26 levels in the plasma of adult TB patients were markedly lower than those of healthy cohorts. We purified recombinant IL-26 from an E. coli expression system using the Ni-NTA resin. Upon stimulations with the recombinant IL-26, human THP1 cells exhibited rapid morphological changes characterized by increased irregular spindle shape and formation of granular structures. Treating THP1 cells with IL-26 can also lead to heightened expressions of CD80, TNF-α, and iNOS but not CD206 and Arg1 in these cells, indicating an M1 macrophage differentiation phenotype. Furthermore, our investigations revealed a dose-dependent escalation of reactive oxygen species production, decreased mitochondrial membrane potential, and enhanced autophagy flux activity in THP1 macrophages following IL-26 treatment. Moreover, our results demonstrated that IL-26 contributed to the elimination of intracellular Mycobacterium tuberculosis via orchestrated ROS production. In conclusion, our findings elucidated the role of IL-26 in the development of tuberculosis and its contributions to intracellular bacilli killing by macrophages through the induction of M1-polarization and ROS production. These insights may have significant implications for understanding the pathogenesis of tuberculosis and developing novel therapeutic strategies.

Challenges and advances in serological and molecular tests to aid leprosy diagnosis.

Leprosy is a neglected chronic infectious disease caused by obligate intracellular bacilli, Mycobacterium leprae and Mycobacterium lepromatosis. Despite multidrug therapy (MDT) success, leprosy accounts for more than 200,000 new cases yearly. Leprosy diagnosis remains based on the dermato-neurologic examination, but histopathology of skin biopsy and bacilloscopy of intradermal scraping are subsidiary diagnostic tests that require expertise and laboratory infrastructure. This minireview summarizes the state of the art of serologic tests to aid leprosy diagnosis, highlighting enzyme-linked immunosorbent assay (ELISA) and point-of-care tests (POCT) biotechnologies. Also, the impact of the postgenomic era on the description of new recombinantly expressed M. leprae-specific protein antigens, such as leprosy Infectious Disease Research Institute (IDRI) diagnostic (LID)-1 is summarized. Highly specific and sensitive molecular techniques to detect M. leprae DNA as the quantitative polymerase chain reaction (qPCR) and the loop-mediated isothermal amplification (LAMP) are briefly reviewed. Serology studies using phenolic glycolipid-I (PGL-I) semi-synthetic antigens, LID-1 fusion antigen, and the single fusion complex natural disaccharide-octyl (NDO)-LID show high sensitivity in multibacillary (MB) patients. However, serology is not applicable to paucibacillary patients, as they have weak humoral response and robust cell-mediated response, requiring tests for cellular biomarkers. Unlike ELISA-based tests, leprosy-specific POCT based on semi-synthetic PGL-I antigens and NDO-LID 1 antigen is easy to perform, cheaper, equipment-free, and can contribute to early diagnosis avoiding permanent incapacities and helping to interrupt M. leprae transmission. Besides its use to help diagnosis of household contacts or at-risk populations in endemic areas, potential applications of leprosy serology include monitoring MDT efficacy, identification of recent infection, especially in young children, as surrogate markers of disease progression to orient adult chemoprophylaxis and as a predictor of type 2 leprosy reactions. Advances in molecular biology techniques have reduced the complexity and execution time of qPCR confirming its utility to help diagnosis while leprosy-specific LAMP holds promise as an adjunct test to detect M. leprae DNA.

Interactive effect of oral anti-hyperglycaemic or anti-hypertensive drugs on the inhibitory and bactericidal activity of first line anti-TB drugs against M. tuberculosis.

Co-existence of life style disorders, like, Diabetes or Hypertension, increases risk of, treatment failure, deaths and developing drug-resistant TB. Concomitant administration of drugs to treat dual/multi-morbidities may alter their effectiveness, in additive/synergistic or adverse/antagonistic manner. We evaluated interactive effect of 7 anti-hyperglycaemic (HG) and 6 anti-hypertensive (HT) drugs on the inhibitory (MICs) and bactericidal (% killing of intracellular bacilli) activities of anti-TB drugs, Isoniazid (INH), Rifampicin (RFM), Ethambutol (EMB) and Streptomycin (STR) against M. tuberculosis. Five anti-HG drugs, namely, Acarbose, Acetohexamide, Glyburide, Repaglinide and Sitagliptin imparted either 'additive' or 'no effect' on the activities (inhibition or % killing) of all the four anti-TB drugs, as evident by their lower FICs (Fractional Inhibitory concentrations) and higher bacterial killing in combination. Metformin and Rosiglitazone, however, exerted adverse effect on the Ethambutol (FICs >2.0). All the six anti-HT drugs, namely, Atenolol, Hydrochlorothiazide, Ramipril, Valsartan, Nifedipine and Verapamil exerted either 'additive'/'synergistic' or 'no effect' on the activities of anti-TB drugs. These findings may help clinicians to select safe and helpful anti-HG or anti-HT drugs for TB patients, if, suffering with diabetes or hypertension like co-morbidities and receiving DOTs (a set regimen for the treatment of TB based on the WHO guidelines).

Eradication of Drug-Tolerant Mycobacterium tuberculosis 2022: Where We Stand.

The lungs of tuberculosis (TB) patients contain a spectrum of granulomatous lesions, ranging from solid and well-vascularized cellular granulomas to avascular caseous granulomas. In solid granulomas, current therapy kills actively replicating (AR) intracellular bacilli, while in low-vascularized caseous granulomas the low-oxygen tension stimulates aerobic and microaerophilic AR bacilli to transit into non-replicating (NR), drug-tolerant and extracellular stages. These stages, which do not have genetic mutations and are often referred to as persisters, are difficult to eradicate due to low drug penetration inside the caseum and mycobacterial cell walls. The sputum of TB patients also contains viable bacilli called differentially detectable (DD) cells that, unlike persisters, grow in liquid, but not in solid media. This review provides a comprehensive update on drug combinations killing in vitro AR and drug-tolerant bacilli (persisters and DD cells), and sterilizing Mycobacterium tuberculosis-infected BALB/c and caseum-forming C3HeB/FeJ mice. These observations have been important for testing new drug combinations in noninferiority clinical trials, in order to shorten the duration of current regimens against TB. In 2022, the World Health Organization, following the results of one of these trials, supported the use of a 4-month regimen for the treatment of drug-susceptible TB as a possible alternative to the current 6-month regimen.

Prevalence of Leprosy in District Buner, Khyber Pakhtunkhwa, Pakistan

Background: Leprosy, commonly referred to as Hansen's disorder, is caused by the obligatory intracellular acid-fast bacilli Mycobacterium leprae and is predominantly transmitted by inhalation macrophages. It causes infection of the skin and nerves, as well as neurological damage due to inflammation. Aim: The current study was conducted to determine the prevalence of leprosy disease in District Buner, Khyber Pakhtunkhwa, Pakistan.During the investigation, we reported different types of leprosy. Methods: The current study investigates 467 patients from 1965-2000 in six Tehsil and 74 from 2000-2021. In the reported five types of Leprosy, Lepromatous Leprosy is more common than other types. Leprosy has five subtypes which 2 are Tuberculoid (TT), Lepromatous Leprosy (LL), and 3 are borderline types Leprosy Borderline tuberculoid (BT), Borderline (BB),borderline lepromatous (BL).

Mmu-miR-25-3p promotes macrophage autophagy by targeting DUSP10 to reduce mycobacteria survival.

The present study aimed to investigate the regulation of miR-25-3p on macrophage autophagy and its effect on macrophage clearance of intracellular Mycobacterium bovis Bacillus Calmette-Guerin (BCG) retention based on the previous findings on the differential expression of exosomal miRNA in macrophages infected with BCG. Through enrichment analysis and Hub gene analysis, key differentially expressed miRNA and its target genes were selected. The targeted binding ability of the screened mmu-miR-25-3p and its predicted target gene DUSP10 was determined through the TargetScan database, and this was further verified by dual luciferase reporter gene assay. mmu-miR-25-3p mimics, mmu-miR-25-3p inhibitor, si-DUSP10, miR-NC,si-NC and PD98059 (ERK Inhibitor) were used to intervene macrophages Raw264.7. Rt-qPCR was used to detect the expression levels of mmu-miR-25-3p and DUSP10 mRNA. Western blot was used to detect the expression levels of DUSP10, LC3-II, p-ERK1/2, beclin1, Atg5 and Atg7. The autophagy flux of macrophage Raw264.7 in each group was observed by confocal laser microscopy, and the expression distribution of DUSP10 and the structure of autophagosomes were observed by transmission electron microscopy. Finally, the intracellular BCG load of macrophage Raw264.7 was evaluated by colony-forming unit (CFU) assay. Bioinformatics analysis filtered and identified the differentially expressed exosomal miRNAs. As a result, mmu-miR-25-3p expression was significantly increased, and dual specificity phosphatase 10 (DUSP10) was predicted as its target gene that was predominantly involved in autophagy regulation. The dual luciferase reporter gene activity assay showed that mmu-miR-25-3p was targeted to the 3'-untranslated region (UTR) of DUSP10. The infection of BCG induced the upregulation of mmu-miR-25-3p and downregulation of DUSP10 in RAW264.7 cells, which further increased the expression of LC3-II and promoted autophagy. Upregulated mmu-miR-25-3p expression decreased the level of DUSP10 and enhanced the phosphorylation of ERK1/2, which in turn upregulated the expression of LC3-II, Atg5, Atg7, and Beclin1. Immuno-electron microscopy, transmission electron microscopy, and autophagic flux analysis further confirmed that the upregulation of mmu-miR-25-3p promotes the autophagy of macrophages after BCG infection. The CFU number indicated that upregulated mmu-miR-25-3p expression decreased the mycobacterial load and accelerated residual mycobacteria clearance. mmu-miR-25-3p promotes the phosphorylation of ERK1/2 by inhibiting the expression of DUSP10, thus enhancing the BCG-induced autophagy of macrophages. These phenomena reduce the bacterial load of intracellular Mycobacterium and facilitate the clearance of residual mycobacteria. mmu-miR-25-3p has great potential as a target for anti-tuberculosis immunotherapy and can be the optimal miRNA loaded into exosomal drug delivery system in future studies.

A Rare Presentation of Scrub Typhus: Multi-Organ Dysfunction [Acute Kidney Injury, Hepatitis] and Acute Respiratory Distress Syndrome

Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi- a gram negative intracellular bacilli. It typically manifests as eschar formation, fever, rash, myalgia and diffuse lymphadenopathy. The clinical severity of scrub typhus ranges from mild fever to a lethal outcome. Complications like sepsis, acute respiratory distress syndrome (ARDS) and multi organ dysfunction syndrome (MODS)--hepatitis, acute kidney injury contribute to the higher mortality rates, which range from 7-30% in untreated cases. Here, we report a case of 35 years old male who presented with complaints of fever, headache, generalised abdominal pain and dry cough for 7 days. The patient was tested positive for Scrub Typhus IgM antibody; and was complicated with MODS- AKI, Hepatitis and ARDS. After extensive intensive care, monitoring and treatment of 15-20 days, the patient recovered. Hence, early diagnosis and prompt treatment aids in decreasing the mortality in such cases.

2,4-Disubstituted pyridine derivatives are effective against intracellular and biofilm-forming tubercle bacilli.

It was recently reported that 4-substituted picolinohydrazonamides carrying hydrophilic cyclic amines, such as morpholine and pyrrolidine, at the end of their thiosemicarbazide chain have potent antimycobacterial activity in vitro at concentrations below 1μg/ml. Here, two selected compounds, 2,4-disubstituted pyridine derivatives 11 and 15, revealed significant bactericidal activity against Mycobacterium tuberculosis localized intracellularly within human macrophages, as well as against biofilm-forming tubercle bacilli. Mutants were selected that were resistant to the investigated compounds at an efficiency similar to that identified in the presence of the first line antituberculosis drug rifampicin. The resistant mutants were viable in the presence of the tested compounds exclusively on solid media. Genome-wide sequencing of the mutants selected in the presence of compound 11 revealed the accumulation of nonsynonymous mutations in the mmpR5 gene encoding a transcriptional repressor of the MmpS5-MmpL5 efflux pump, whose upregulation has been associated with bedaquiline resistance. The depletion of MmpR5 in wild-type M. tuberculosis using CRISPR-Cas9 technology increased the resistance of this strain to compound 11. Mass spectrometry-based proteomics (LC-MS/MS) of wild-type tubercle bacilli growing in subinhibitory concentrations of compounds 11 or 15 revealed 15 overproduced proteins not detectable in the control cells, including virulence-related proteins.

Intramacrophage potential of a tetrahydropyridine: A promising compound in combating Mycobacterium tuberculosis

Due to several obstacles in treating tuberculosis (TB), the search for new therapeutic alternatives remains a global priority. The nitrogenous heterocyclic compounds are promising in searching for new anti-Mycobacterium tuberculosis molecules, and our previous results highlight the potential of tetrahydropyridines. After exploring the antimycobacterial potential and putative mechanism of action of a tetrahydropyridine derivative (NUNL02), we seek to measure the oxidative stress caused by NUNL02 inside the extracellular replicating M. tuberculosis since it could be the reason for the NUNL02 bactericidal effect against replicating and starved M. tuberculosis; and to evaluate the anti-M. tuberculosis activity of NUNL02 against the intracellular bacillus (even combined with an anti-TB drug) to explore the potential of this tetrahydropyridine as a promising adjuvant for TB therapy. Briefly, we assessed the activity of NUNL02 against the H37Rv strain and evaluated the combination of NUNL02 and rifampicin (RIF), at previously defined subinhibitory concentrations, against intramacrophage M. tuberculosis. NUNL02, in addition to promote the oxidative stress inside the extracellular replicating M. tuberculosis as a possible indirect mechanism of action, also presented bactericidal potential as promising as RIF against intracellular bacilli. Thus, our findings reinforce NUNL02 as a promising scaffold for the development of new options for TB.

Infecciones por Rickettsia y fiebre Q

Rickettsioses are prevalent zoonoses worldwide. They are caused by a group of gram-negative obligate intracellular coccobacilli and bacilli. In the biological cycle, various mammals which act as reservoirs and arthropods which act as vectors play a role. In addition to classical symptoms such as Mediterranean spotted fever and murine typhus, new clinical entities have appeared in Spain in recent years. They cause initial nonspecific symptoms that are very similar; later, other, more specific manifestations of each type of rickettsia appear. Q fever is another zoonosis with a worldwide distribution caused by Coxiella burnetti. The main mechanism of transmission is through aerosols, although other mechanisms of transmission have been described. The infection presents in the form of acute or primary symptoms and, in a small percentage of cases, as chronic or persistent disease. The etiological diagnosis of rickettsioses and Q fever is commonly made during convalescence by means of specific serological tests. When faced with clinical suspicion, empirical treatment should be started. Doxycycline is the drug of choice for nearly all of these infections.

Fighting MDR-Klebsiella pneumoniae Infections by a Combined Host- and Pathogen-Directed Therapeutic Approach.

Klebsiella pneumoniae is an opportunistic pathogen that is very difficult to treat mainly due to its high propensity to acquire complex resistance traits. Notably, multidrug resistance (MDR)-Klebsiella pneumoniae (KP) infections are responsible for 22%–72% of mortality among hospitalized and immunocompromised patients. Although treatments with new drugs or with combined antibiotic therapies have some degree of success, there is still the urgency to investigate and develop an efficient approach against MDR-KP infections. In this study, we have evaluated, in an in vitro model of human macrophages, the efficacy of a combined treatment consisting of apoptotic body-like liposomes loaded with phosphatidylinositol 5-phosphate (ABL/PI5P) and φBO1E, a lytic phage specific for the major high-risk clone of KPC-positive MDR-KP. Results show that ABL/PI5P did not affect in a direct manner KKBO-1 viability, being able to reduce only the intracellular KKBO-1 bacterial load. As expected, φBO1E was effective mainly on reducing extracellular bacilli. Importantly, the combination of both treatments resulted in a simultaneous reduction of both intracellular and extracellular bacilli. Moreover, the combined treatment of KKBO-1-infected cells reduced proinflammatory TNF-α and IL-1β cytokines and increased anti-inflammatory TGF-β cytokine production. Overall, our data support the therapeutic value of a combined host- and pathogen-directed therapy as a promising approach, alternative to single treatments, to simultaneously target intracellular and extracellular pathogens and improve the clinical management of patients infected with MDR pathogens such as MDR-KP.

Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection.

ABSTRACTMycobacterium abscessus is the etiological agent of severe pulmonary infections in vulnerable patients, such as those with cystic fibrosis (CF), where it represents a relevant cause of morbidity and mortality. Treatment of pulmonary infections caused by M. abscessus remains extremely difficult, as this species is resistant to most classes of antibiotics, including macrolides, aminoglycosides, rifamycins, tetracyclines, and β-lactams. Here, we show that apoptotic body like liposomes loaded with phosphatidylinositol 5-phosphate (ABL/PI5P) enhance the antimycobacterial response, both in macrophages from healthy donors exposed to pharmacological inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) and in macrophages from CF patients, by enhancing phagosome acidification and reactive oxygen species (ROS) production. The treatment with liposomes of wild-type as well as CF mice, intratracheally infected with M. abscessus, resulted in about a 2-log reduction of pulmonary mycobacterial burden and a significant reduction of macrophages and neutrophils in bronchoalveolar lavage fluid (BALF). Finally, the combination treatment with ABL/PI5P and amikacin, to specifically target intracellular and extracellular bacilli, resulted in a further significant reduction of both pulmonary mycobacterial burden and inflammatory response in comparison with the single treatments. These results offer the conceptual basis for a novel therapeutic regimen based on antibiotic and bioactive liposomes, used as a combined host- and pathogen-directed therapeutic strategy, aimed at the control of M. abscessus infection, and of related immunopathogenic responses, for which therapeutic options are still limited.IMPORTANCE Mycobacterium abscessus is an opportunistic pathogen intrinsically resistant to many antibiotics, frequently linked to chronic pulmonary infections, and representing a relevant cause of morbidity and mortality, especially in immunocompromised patients, such as those affected by cystic fibrosis. M. abscessus-caused pulmonary infection treatment is extremely difficult due to its high toxicity and long-lasting regimen with life-impairing side effects and the scarce availability of new antibiotics approved for human use. In this context, there is an urgent need for the development of an alternative therapeutic strategy that aims at improving the current management of patients affected by chronic M. abscessus infections. Our data support the therapeutic value of a combined host- and pathogen-directed therapy as a promising approach, as an alternative to single treatments, to simultaneously target intracellular and extracellular pathogens and improve the clinical management of patients infected with multidrug-resistant pathogens such as M. abscessus.

Mycobacterium tuberculosis Rv0580c Impedes the Intracellular Survival of Recombinant Mycobacteria, Manipulates the Cytokines, and Induces ER Stress and Apoptosis in Host Macrophages via NF-κB and p38/JNK Signaling.

The Mycobacterium tuberculosis (M. tb) genome encodes a large number of hypothetical proteins, which need to investigate their role in physiology, virulence, pathogenesis, and host interaction. To explore the role of hypothetical protein Rv0580c, we constructed the recombinant Mycobacterium smegmatis (M. smegmatis) strain, which expressed the Rv0580c protein heterologously. We observed that Rv0580c expressing M. smegmatis strain (Ms_Rv0580c) altered the colony morphology and increased the cell wall permeability, leading to this recombinant strain becoming susceptible to acidic stress, oxidative stress, cell wall-perturbing stress, and multiple antibiotics. The intracellular survival of Ms_Rv0580c was reduced in THP-1 macrophages. Ms_Rv0580c up-regulated the IFN-γ expression via NF-κB and JNK signaling, and down-regulated IL-10 expression via NF-κB signaling in THP-1 macrophages as compared to control. Moreover, Ms_Rv0580c up-regulated the expression of HIF-1α and ER stress marker genes via the NF-κB/JNK axis and JNK/p38 axis, respectively, and boosted the mitochondria-independent apoptosis in macrophages, which might be lead to eliminate the intracellular bacilli. This study explores the crucial role of Rv0580c protein in the physiology and novel host-pathogen interactions of mycobacteria.

Harmful Effects of Granulocytic Myeloid-Derived Suppressor Cells on Tuberculosis Caused by Hypervirulent Mycobacteria.

The role of myeloid-derived suppressor cells (MDSCs) in patients with severe tuberculosis who suffer from uncontrolled pulmonary inflammation caused by hypervirulent mycobacterial infection remains unclear. This issue was addressed using C57BL/6 mice infected with highly virulent Mycobacterium bovis strain MP287/03. CD11b+GR1int population increased in the bone marrow, blood and lungs during advanced disease. Pulmonary CD11b+GR1int (Ly6GintLy6Cint) cells showed granularity similar to neutrophils and expressed immature myeloid cell markers. These immature neutrophils harbored intracellular bacilli and were preferentially located in the alveoli. T-cell suppression occurred concomitantly with CD11b+GR1int cell accumulation in the lungs. Furthermore, lung and bone marrow GR1+ cells suppressed both T-cell proliferation and interferon γ production in vitro. Anti-GR1 therapy given when MDSCs infiltrated the lungs prevented expansion and fusion of primary pulmonary lesions and the development of intragranulomatous caseous necrosis, along with increased mouse survival and partial recovery of T-cell function. Lung bacterial load was reduced by anti-GR1 treatment, but mycobacteria released from the depleted cells proliferated extracellularly in the alveoli, forming cords and clumps. Granulocytic MDSCs massively infiltrate the lungs during infection with hypervirulent mycobacteria, promoting bacterial growth and the development of inflammatory and necrotic lesions, and are promising targets for host-directed therapies.

Patterns of Mycobacterium avium-intracellulare complex infection in duodenal endoscopic biopsies in HIV/AIDS patients

Mycobacterium avium-intracellulare complex (MAIC) is a nontuberculous opportunistic infection in immunocompromised patients. Involvement of the gastrointestinal tract (GIT) is usually part of a disseminated disease in AIDS patients with a low CD4 count, however with standard antiretroviral therapy (ART), a localized presentation is more likely. It can affect any part of the GIT, mostly the duodenum and typically as patches. Incomplete or refractory ART for HIV-strains, therapy-related side effects, noncompliant or incomplete treatment to previous MAIC infections, superimposed complications and comorbid opportunistic infections may result in atypical clinical, endoscopic and histopathologic manifestations. We performed a retrospective review study retrieving cases of MAIC in duodenal endoscopic biopsy. We found five cases of MAIC in HIV/AIDS patients. They were males with an average age of 40-years. They showed different histopathologic features, variable patterns of MAIC-histiocytic infiltrates, and varying intensity of intracellular acid-fast positive bacilli. Enterocytes vacuolization and transepithelial elimination were also observed. Three cases were associated with cytomegalovirus and cryptococcal infections. A case was complicated by lymphangiectasia-associated protein-losing enteropathy. Initially, three cases were morphologically missed. Ziehl-Neelsen stain helped reach the correct diagnosis. Pathologists have an important role in patients' management by guiding clinicians to the correct diagnosis. Pathologists should be aware of these different histopathologic manifestations, their potential pitfalls, look for certain helpful clues complemented with multiple levels and special stains. In particular, AFB stains are mandatory in all mucosal biopsy specimens from HIV/AIDS patients regardless of their appearances.