Intracellular Killing Of Bacteria Research Articles

Bosutinib Stimulates Macrophage Survival, Phagocytosis, and Intracellular Killing of Bacteria.

Host-acting compounds are emerging as potential alternatives to combating antibiotic resistance. Here, we show that bosutinib, an FDA-approved chemotherapeutic for treating chronic myelogenous leukemia, does not possess any antibiotic activity but enhances macrophage responses to bacterial infection. In vitro, bosutinib stimulates murine and human macrophages to kill bacteria more effectively. In a murine wound infection with vancomycin-resistant Enterococcus faecalis, a single intraperitoneal bosutinib injection or multiple topical applications on the wound reduce the bacterial load by approximately 10-fold, which is abolished by macrophage depletion. Mechanistically, bosutinib stimulates macrophage phagocytosis of bacteria by upregulating surface expression of bacterial uptake markers Dectin-1 and CD14 and promoting actin remodeling. Bosutinib also stimulates bacterial killing by elevating the intracellular levels of reactive oxygen species. Moreover, bosutinib drives NF-κB activation, which protects infected macrophages from dying. Other Src kinase inhibitors such as DMAT and tirbanibulin also upregulate expression of bacterial uptake markers in macrophages and enhance intracellular bacterial killing. Finally, cotreatment with bosutinib and mitoxantrone, another chemotherapeutic in clinical use, results in an additive effect on bacterial clearance in vitro and in vivo. These results show that bosutinib stimulates macrophage clearance of bacterial infections through multiple mechanisms and could be used to boost the host innate immunity to combat drug-resistant bacterial infections.

Ex vivo Platforms to Study the Primary and Recall Immune Responses to Intracellular Mycobacterial Pathogens and Peptide-Based Vaccines.

Progress in the study of the immune response to pathogens and candidate vaccines has been impeded by limitations in the methods to study the functional activity of T-cell subsets proliferating in response to antigens processed and presented by antigen presenting cells (APC). As described in this review, during our studies of the bovine immune response to a candidate peptide-based vaccine and candidate rel deletion mutants in Mycobacterium avium paratuberculosis (Map) and Mycbacterium bovis (BCG), we developed methods to study the primary and recall CD4 and CD8 T-cell responses using an ex vivo platform. An assay was developed to study intracellular killing of bacteria mediated by CD8 T cells using quantitative PCR to distinguish live bacteria from dead bacteria in a mixed population of live and dead bacteria. Through use of these assays, we were able to demonstrate vaccination with live rel Map and BCG deletion mutants and a Map peptide-based vaccine elicit development of CD8 cytotoxic T cells with the ability to kill intracellular bacteria using the perforin-granzyme B pathway. We also demonstrated tri-directional signaling between CD4 and CD8 T cells and antigen-primed APC is essential for eliciting CD8 cytotoxic T cells. Herein, we describe development of the assays and review progress made through their use in the study of the immune response to mycobacterial pathogens and candidate vaccines. The methods obviate some of the major difficulties encountered in characterizing the cell-mediated immune response to pathogens and development of attenuated and peptide-based vaccines.

One-step light-up metabolic probes for in situ discrimination and killing of intracellular bacteria

TPEPy-Ala and TPAPy-Kdo with metabolic moieties can be directly incorporated into the bacterial cell envelopes and light up intracellular bacteria. Additionally, the metabolic probes can effectively eliminate labeled bacteria in situ with minimal host cell cytotoxicity via photodyanmic therapy.

Improvement of anti-biofilm activities via co-delivery of curcumin and gentamicin in lipid-polymer hybrid nanoparticle

Pseudomonas aeruginosa is the most common pathogen that causes chronic lung infections and recurrence of the disease in cystic fibrosis patients by hiding inside cells and biofilm matrix. Herein, we developed gentamicin and curcumin-loaded lipid-polymer hybrid nanoparticle- (termed CG-HNPs) to evaluate in vitro activities against biofilm-embedded P. aeruginosa and compared with lipid nanoparticles containing the same drugs (CG-Lip). The nanoparticles were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), fluorescence spectroscopy, and ultraviolet-visible (UV-vis) spectroscopy, which demonstrated that HNPs with a diameter of approximately 340 nm were uniform. The optimal CG-HNPs formulation illustrated high encapsulation (∼70%) and controlled release characteristics (gradually released in 72 h). The antibacterial activities of generated nanoparticles are maintained against planktonic and biofilm bacteria and it is effective in damage established biofilms. Besides, HNPs were biocompatible and nontoxic to J774 and HFF cell lines and uptake by the macrophages (J774), which facilitated the killing of intracellular bacteria in macrophages. These results introduced CG-HNPs as a promising antibacterial agent for the treatment of chronic infections and intracellular bacteria due to excellent antibacterial activity.

Study on Significance of Receptor Targeting in Killing of Intracellular Bacteria with Membrane‐Impermeable Antibiotics

AbstractWater‐soluble antibiotics are largely excluded from therapy of intracellular infections, such as Shigella spp., Listeria spp., or Salmonella spp., due to their inability to permeate mammalian cell membrane. Here, the authors study if targeting offers an advantage to deliver killing doses of membrane‐impermeable antibiotics intracellularly to infected cells. Mannose‐decorated liposomes, loaded with gentamicin, are fabricated to target mannose receptor, a recognition system of (infected) macrophages. Designing a family of liposomes with varying surface presentation of mannose ligand, the authors show a clear dependence of cellular internalization on the ligand surface presentation. Significantly for the killing of intracellular bacteria, the study demonstrates internalization of mannosylated liposomes by the entire population of macrophages, both Salmonella‐infected and non‐infected, resulting in an efficient treatment of intracellular infection. This contrasts with non‐targeted liposomes, where internalization does not occur by a substantial subpopulation of infected cells. The study points to the significance of targeted delivery of antibiotics for treatment of intracellular infections.

Administration of GDF3 Into Septic Mice Improves Survival via Enhancing LXRα-Mediated Macrophage Phagocytosis.

The defective eradication of invading pathogens is a major cause of death in sepsis. As professional phagocytic cells, macrophages actively engulf/kill microorganisms and play essential roles in innate immune response against pathogens. Growth differentiation factor 3 (GDF3) was previously implicated as an important modulator of inflammatory response upon acute sterile injury. In this study, administration of recombinant GDF3 protein (rGDF3) either before or after CLP surgery remarkably improved mouse survival, along with significant reductions in bacterial load, plasma pro-inflammatory cytokine levels, and organ damage. Notably, our in vitro experiments revealed that rGDF3 treatment substantially promoted macrophage phagocytosis and intracellular killing of bacteria in a dose-dependent manner. Mechanistically, RNA-seq analysis results showed that CD5L, known to be regulated by liver X receptor α (LXRα), was the most significantly upregulated gene in rGDF3-treated macrophages. Furthermore, we observed that rGDF3 could promote LXRα nuclear translocation and thereby, augmented phagocytosis activity in macrophages, which was similar as LXRα agonist GW3965 did. By contrast, pre-treating macrophages with LXRα antagonist GSK2033 abolished beneficial effects of rGDF3 in macrophages. In addition, rGDF3 treatment failed to enhance bacteria uptake and killing in LXRα-knockout (KO) macrophages. Taken together, these results uncover that GDF3 may represent a novel mediator for controlling bacterial infection.

Everolimus-induced effector mechanism in macrophages and survivability of Erdman, CDC1551 and HN878 strains of Mycobacterium tuberculosis infection.

With a disease as widespread and destructive as tuberculosis, more effective drugs and healthcare strategies, in addition to the current antibiotics regimen, are crucial for the enhanced well-being of millions of people suffering from the disease. Host-directed therapy is a new and emerging concept in treating chronic infectious diseases, such as tuberculosis. Repurposing of anti-cancer drugs, such as everolimus, may be an effective way to supplement the standard antibiotic treatment. Individuals with type 2 diabetes are increasingly susceptible to co-morbidities and co-infections including Mycobacterium tuberculosis, the causative agent of tuberculosis. We demonstrated in this study that in vitro everolimus treatment of granulomas from individuals with type 2 diabetes caused significant reduction in the viability of Mycobacterium tuberculosis.Further investigations revealed the effects of everolimus in targeting foamy macrophages, a macrophage phenotype that forms around granulomas, and is characterized by a higher lipid accumulation inside the cells. These foamy macrophages are thought to harbor dormant bacilli, which are potential sources of disease reactivation. Therefore, blocking foamy macrophage formation would help better killing of intracellular bacteria. Here, we report the potential of everolimus treatment to downregulate lipid content within the foamy macrophages of in vitro granulomas, thus leading to a potential decrease in the number of foamy macrophages and a more robust response to Mycobacterium tuberculosis.

The effect of BCG vaccination on macrophage phenotype in a mouse model of intranasal Mycobacterium bovis challenge

In order to develop improved vaccinations against tuberculosis, it is essential to understand the effect of vaccination on the immune response, and to overcome the mechanisms by which mycobacteria regulate this immune response. In this study, we examine the effect of intradermal vaccination with Mycobacterium bovis bacille Calmette-Guèrin on macrophage phenotype following intranasal challenge with virulent Mycobacterium bovis. Preserved lung tissues used in the present study were obtained from a previous vaccination trial in BALB/c mice. Vaccinated mice showed less extensive pulmonary lesions along with a significant decrease in bacterial lung burden when compared to control mice. Immunohistochemical markers of classically activated macrophages (iNOS) and alternatively activated macrophages (Arg1, FIZZ1) were applied to lung sections. Vaccination led to a statistically significant decrease in the number of Arg1+ macrophages. The presence of macrophages that expressed Arginase 1 in pulmonary lesions was much smaller than the presence of macrophages expressing iNOS. The low presence of Arg1+ macrophages induced by vaccination may be caused by Th1 polarization and may reduce alternative activation of macrophages, with an overall more effective intracellular killing of bacteria.

Gentamicin decorated phosphatidylcholine-chitosan nanoparticles against biofilms and intracellular bacteria

Biofilms and intracellular bacteria often cause a series of overwhelming public health issues due to their strong drug resistance. Hence, chitosan nanoparticles (CS NPs), phosphatidylcholine and gentamicin were used to synthesize a novel nanodrug delivery system (GPC NPs). Dynamic light scattering (DLS) demonstrated that the surface zeta-potential of GPC NPs was −19.5 mV. The morphology of GPC NPs was observed by scanning electron microscopy (SEM). The gentamicin adsorption and release behaviors of GPC NPs were also investigated. The GPC NPs could effectively damage and remove the biofilm formed by pathogens through permeation of the antibiotic into the biofilm. In addition, the nanoparticles were readily engulfed by macrophages which facilitated the killing of intracellular bacteria and had neglectable cytotoxicity. Our study indicated that GPC NPs could be used as a promising nanoantibacterial agent against biofilms and intracellular bacteria.

Diagnostic value of CD64 in early detection of neonatal sepsis

Early diagnosis of neonatal sepsis is challenging because clinical picture is not specific and hard to be discriminated from that of noninfectious causes. The gold standard tool for diagnosis of neonatal sepsis is blood culture. Cluster of differentiation 64 (CD64) is a type of integral membrane glycoprotein known as Fc receptor that binds monomeric IgG with high affinity. Stimulation via CD64 leads to elevated neutrophil capacity for phagocytosis and intracellular killing of bacteria. The aim of this study was to evaluate the role of neutrophil CD64 expression for the identification of sepsis in neonates and to state its relationship with C-reactive protein (CRP). This study included 74 neonates divided into three groups: group 1, documented sepsis (26 patients with positive bacterial culture); group 2, clinical sepsis (25 patients without culture-proven sepsis with the presence of two or more clinical symptoms or signs of sepsis); and group 3, control group (twenty-three healthy neonates). CD64 was measured at admission using enzyme-linked immunosorbent assay (ELISA) kits. A cutoff value of ≥ 535.6 pg/dl can predict neonatal sepsis with 96.1% sensitivity and 91.3% specificity. Serum CD64 was significantly higher in documented and clinical sepsis groups than in control group (p = 0.001, 0.009), respectively. Also serum Cd64 was significantly higher in documented than in clinical sepsis group (p = 0.001).A positive correlation was found between CRP and CD64. This study revealed that serum levels of CD64 can be a sensitive marker for early detection of neonatal sepsis.

P021 The influence of short-chain fatty acids produced by commensal bacteria on macrophage phenotype

Abstract Background Macrophages are dynamic immune cells that react to environmental signals to adapt to their environment. Among this diverse array of signals are multiple bacterially derived metabolites including short-chain fatty acids (SCFAs) in the gastrointestinal tract. It has been previously shown that butyrate, one of several SCFAs produced by bacteria, is able to drive an antimicrobial phenotype in macrophages. However, it is still unknown which bacterial species are able to drive this impact and whether this effect can be recapitulated in vivo. The identification of a common molecular signature that drives this phenotype may allow us to easily identify bacteria with this beneficial phenotype and give insight into potential therapeutic strains. Methods Monocytes were differentiated into macrophages in the presence of SFCAs or bacterial supernatants from a wide range of gut commensals isolated from healthy human donors. RNA was isolated post-differentiation and gene expression screened using a defined gene panel using the Fluidgm Biomark system. In addition, cytokines present in the supernatant were measured by ELISA and the ability of the macrophages to kill intracellular bacteria determined. Currently, we are carrying out the same procedure using bone marrow-derived macrophages (BMDM) from mice in order to expand this work flow into the mouse system so that we can determine if bacteria identified can elicit their impact in vivo. Results We have screened numerous SCFAs alone and in combination in the bacterial killing assay to determine how these are able to influence macrophage phenotype. From these experiments, we have repeated the earlier observation that butyrate can increase macrophage killing of intracellular bacteria. In addition, we have identified four bacterial isolates that induce significantly increased microbial killing in macrophages. This is associated with the production of butyrate, acetate and formate. Using RNA from these different conditions, we are in the process of determining if there is a common molecular pathway by which this increased antimicrobial phenotype is driven. Conclusion Both SCFAs and SCFA producing bacteria are able to induce an antimicrobial phenotype in macrophages that increased the ability of these cells to kill intracellular pathogens. Identification of a common signature that drives these changes could help to screen bacterial isolates for this beneficial trait. Ongoing work hopes to identify such a signature, to enable us to screen large numbers of commensals using high-throughput molecular methods. This can then be verified using both in vitro and in vivo techniques.

Immunological Impacts of Diabetes on the Susceptibility of Mycobacterium tuberculosis.

The interaction between diabetes and major world infections like TB is a major public health concern because of rapidly rising levels of diabetes. The dual burden of tuberculosis (TB) and diabetes mellitus (DM) has become a major global public health problem. Diabetes mellitus is a major risk factor for the development of active and latent tuberculosis. Immune mechanisms contributing to the increased susceptibility of diabetic patients to TB are due to the defects in bacterial recognition, phagocytic activity, and cellular activation which results in impaired production of chemokines and cytokines. The initiation of adaptive immunity is delayed by impaired antigen-presenting cell (APC) recruitment and function in hyperglycemic host, which results in reduced frequencies of Th1, Th2, and Th17 cells and its secretion of cytokines having a great role in activation of macrophage and inflammatory response of tuberculosis. In addition, impaired immune response and killing of intracellular bacteria potentially increase bacterial load, chronic inflammation, and central necrosis that facilitate bacterial dissemination and miliary tuberculosis. Understanding of the immunological and biochemical basis of TB susceptibility in diabetic patients will tell us the rational development of implementation and therapeutic strategies to alleviate the dual burden of the diseases. Therefore, the aim of this review was focused on the association between diabetes and tuberculosis, focusing on epidemiology, pathogenesis, and immune dysfunction in diabetes mellitus, and its association with susceptibility, severity, and treatment outcome failure to tuberculosis.

Glucosamine/L-lactide copolymers as potential carriers for the development of a sustained rifampicin release system using Mycobacterium smegmatis as a tuberculosis model

The present study aims at developing a new, ultrafine particle-based efficient antibiotic delivery system for the treatment of tuberculosis. The carrier material to make the rifampicin (RIF)-loaded particles is a low molecular weight star-shaped polymer produced from glucosamine (core building unit) and L-lactide (GluN-LLA). Particles were made via electrohydrodynamic atomization. Prolonged release (for up to 14 days) of RIF from these particles is reported. Drug release data fits the Korsmeyer-Peppas equation, which suggests the occurrence of a modified diffusion-controlled RIF release mechanism in vitro and is also supported by differential scanning calorimetry and drug leaching tests. Cytotoxicity tests on Mycobacterium smegmatis showed that antibiotic-free GluN-LLA and polylactides (PLA) particles (reference materials) did not show any significant anti-bacterial activity. The minimum inhibitory concentration and minimum bactericidal concentration values obtained for RIF-loaded particles showed 2- to 4-fold improvements in the anti-bacterial activity relative to the free drug. Cytotoxicity tests on macrophages indicated that cell death correlates with an increase of particle concentration but is not significantly affected by material type or particle size. Confocal microscopy was used to track internalization and localization of particles in the macrophages. The uptake of GluN-LLA particles is higher than those of their PLA counterparts. In addition, after phagocytosis, the GluN-LLA particles stayed in the cytoplasm and showed favorable long-term drug release behavior, which facilitated the killing of intracellular bacteria when compared to free RIF. The present studies suggest that these drug carrier materials are potentially very attractive candidates for the development of high-payload, sustained-release antibiotic/resorbable polymer particle systems for treating bacterial lung infections.

Aspherical and Spherical InvA497-Functionalized Nanocarriers for Intracellular Delivery of Anti-Infective Agents

PurposeThe objective of this work was to evaluate the potential of polymeric spherical and aspherical invasive nanocarriers, loaded with antibiotic, to access and treat intracellular bacterial infections.MethodsAspherical nanocarriers were prepared by stretching of spherical precursors, and both aspherical and spherical nanocarriers were surface-functionalized with the invasive protein InvA497. The relative uptake of nanocarriers into HEp-2 epithelial cells was then assessed. Nanocarriers were subsequently loaded with a preparation of the non-permeable antibiotic gentamicin, and tested for their ability to treat HEp-2 cells infected with the enteroinvasive bacterium Shigella flexneri.ResultsInvA497-functionalized nanocarriers of both spherical and aspherical shape showed a significantly improved rate and extent of uptake into HEp-2 cells in comparison to non-functionalized nanocarriers. Functionalized and antibiotic-loaded nanocarriers demonstrated a dose dependent killing of intracellular S. flexneri. A slight but significant enhancement of intracellular bacterial killing was also observed with aspherical as compared to spherical functionalized nanocarriers at the highest tested concentration.ConclusionsInvA497-functionalized, polymer-based nanocarriers were able to efficiently deliver a non-permeable antibiotic across host cell membranes to affect killing of intracellular bacteria. Functionalized nanocarriers with an aspherical shape showed an interesting future potential for intracellular infection therapy.

Structure and function of a novel chitin-uptake channel by marine Vibrio species

The present study aims at developing a new, ultrafine particle-based efficient antibiotic delivery system for the treatment of tuberculosis. The carrier material to make the rifampicin (RIF)-loaded particles is a low molecular weight star-shaped polymer produced from glucosamine (core building unit) and L-lactide (GluN-LLA). Particles were made via electrohydrodynamic atomization. Prolonged release (for up to 14 days) of RIF from these particles is reported. Drug release data fits the Korsmeyer-Peppas equation, which suggests the occurrence of a modified diffusion-controlled RIF release mechanism in vitro and is also supported by differential scanning calorimetry and drug leaching tests. Cytotoxicity tests on Mycobacterium smegmatis showed that antibiotic-free GluN-LLA and polylactides (PLA) particles (reference materials) did not show any significant anti-bacterial activity. The minimum inhibitory concentration and minimum bactericidal concentration values obtained for RIF-loaded particles showed 2- to 4-fold improvements in the anti-bacterial activity relative to the free drug. Cytotoxicity tests on macrophages indicated that cell death correlates with an increase of particle concentration but is not significantly affected by material type or particle size. Confocal microscopy was used to track internalization and localization of particles in the macrophages. The uptake of GluN-LLA particles is higher than those of their PLA counterparts. In addition, after phagocytosis, the GluN-LLA particles stayed in the cytoplasm and showed favorable long-term drug release behavior, which facilitated the killing of intracellular bacteria when compared to free RIF. The present studies suggest that these drug carrier materials are potentially very attractive candidates for the development of high-payload, sustained-release antibiotic/resorbable polymer particle systems for treating bacterial lung infections.

Cxxc Finger Protein 1 Positively Regulates GM-CSF-Derived Macrophage Phagocytosis Through Csf2rα-Mediated Signaling.

Macrophages have a defensive function against bacteria through phagocytosis and the secretion of cytokines. Histone modifications play an essential role in macrophage functions. Here, we report that Cxxc finger protein 1 (CFP1), a key component of the SETD1 histone methyltransferase complex, promoted the phagocytic and bactericidal activity of GM-CSF-derived macrophages. CFP1-deficient mice were more susceptible to bacterial infection due to the decreased expression of Csf2rα, a subunit of the GM-CSF receptor essential for inflammation and alveolar macrophage development, through the loss of H3K4 modifications in the promoter of the Csf2rα gene. In addition, the lung tissues of CFP1-deficient mice exhibited spontaneous inflammatory symptoms, including both the infiltration of inflammatory cells and the accumulation of surfactant phospholipids and proteins. Furthermore, we showed that Csf2rα and PU.1 can partially rescue the defects in phagocytosis and in the intracellular killing of bacteria. Collectively, our data highlight the importance of CFP1 in the phagocytic and bactericidal activity of macrophages.

Mycobacterium tuberculosis-Infected Hematopoietic Stem and Progenitor Cells Unable to Express Inducible Nitric Oxide Synthase Propagate Tuberculosis in Mice.

Persistence of Mycobacterium tuberculosis within human bone marrow stem cells has been identified as a potential bacterial niche during latent tuberculosis. Using a murine model of tuberculosis, we show here that bone marrow stem and progenitor cells containing M. tuberculosis propagated tuberculosis when transferred to naive mice, given that both transferred cells and recipient mice were unable to express inducible nitric oxide synthase, which mediates killing of intracellular bacteria via nitric oxide. Our findings suggest that bone marrow stem and progenitor cells containing M. tuberculosis propagate hallmarks of disease if nitric oxide-mediated killing of bacteria is defective.

Extracellular Adenosine Triphosphate Protects Against Sepsis by Enhancing the Intracellular Killing of Bacteria

Extracellular Adenosine Triphosphate Protects Against Sepsis by Enhancing the Intracellular Killing of Bacteria

Saliva induces expression of antimicrobial peptides and promotes intracellular killing of bacteria in keratinocytes by epidermal growth factor receptor transactivation.

Wounds in the oral cavity, constantly exposed to both saliva and bacteria, heal quickly without infection. Furthermore, during licking of skin wounds, saliva promotes wound healing and plays a role in keeping the wound free of infection. To investigate whether saliva induces expression of antimicrobial peptides (AMPs) in human epidermal keratinocytes and whether saliva promotes clearance of intracellular bacteria in these cells. Expression of AMPs was investigated in the oral mucosa and ex vivo injured skin by immunohistochemistry. Human beta-defensin-3 expression was investigated in epidermal keratinocytes after saliva stimulation, using real-time polymerase chain reaction and immunofluorescence. We found higher expression of AMPs in the oral mucosa than in the epidermis. Saliva accelerated the injury-induced expression of AMPs in human skin ex vivo and was a potent inducer of the expression of AMPs in epidermal keratinocytes. The expression of AMPs was induced by metalloproteinase-dependent epidermal growth factor receptor (EGFR) transactivation mediated by a salivary lipid. Saliva increased the intracellular clearance of Staphylococcus aureus in keratinocytes through EGFR activation. These findings suggest a previously unreported role of saliva in innate immunity and demonstrate for the first time that saliva induces gene expression in epidermal keratinocytes.

Clinical, Immunological, and Molecular Findings of Patients with p47phox Defect Chronic Granulomatous Disease (CGD) in Indian Families.

Chronic granulomatous disease (CGD) is a group of inherited disorder of phagocytes, resulting from mutations in the components of the NADPH oxidase complex. Reduced or absent oxygen radical synthesis seen in these patients leads to impaired killing of intracellular bacteria and fungi. CGD clinically presents with recurrent and life-threatening infections as well as granulomatous inflammatory responses. p47phox encoded by the NCF1 gene is the most common autosomal recessive form of CGD which is often clinically milder. Here, we are presenting the data on clinical and immunological findings in 21 Indian patients with Del GT mutation in the NCF1 gene. Diagnosis of these patients was based on detailed clinical evaluation, measurement of respiratory burst activity by nitro blue tetrazolium and dihydrorhodamine-1,2,3 assay, expression of p47phox by flow cytometry, and molecular confirmation by GeneScan method. Seventeen male and four female patients with median age of onset of 1year ranging from 1.5months to 6years were included in the study. Sixty-two percent (13 out of 21) of patients belonged to a consanguineous marriage with only one family having a history of a previous sibling death. Significant variability in clinical presentation was observed in spite of identical genetic defect ranging from asymptomatic to very severe presentation leading to early death or requiring transplantation. However, none of these patients showed difference in immunological parameters to account for this variability. Thus, this study highlights the phenotypic heterogeneity seen in these patients with Del GT mutation in the NCF1 gene and its implication in management of these patients.