Complement C1q is involved in the activation of membrane attack complexes, regulation of bacterial infectious inflammation, and apoptosis through overexpression in primary cells of silver pomfret (Pampus argenteus) in vitro.

The complement system is pivotal in innate immune defense, with Complement 1qb (C1qb) playing a key role in recognizing immune complexes and initiating the classical pathway. In this research, we cloned the full-length cDNA of silver pomfret (Pampus argenteus) c1qb and demonstrated its role in mediating defense responses against Nocardia seriolae (N. seriolae) infection, which notably causes significant economic losses in the aquaculture industry. Our investigation revealed that N. seriolae infection led to tissue damage in fish bodies, as observed in tissue sections. Subsequent analysis of differential genes (DEGs) in the transcriptome highlighted genes linked to apoptosis and inflammation. Through experiments involving overexpression and interference of c1qb in vitro, we confirmed that c1qb could suppress N. seriolae-induced apoptosis and inflammation. Moreover, overexpression of c1qb hindered N. seriolae invasion, and the purified and replicated C1qb protein displayed antimicrobial properties. Additionally, our study unveiled that overexpression of c1qb might stimulate the expression of membrane attack complexes (MAC), potentially enhancing opsonization and antibacterial effects. In conclusion, our findings offer valuable insights into the immune antibacterial mechanisms of c1qb and contribute to the development of strategies for controlling N. seriolae.

Molecular characterization and functional analysis of IL-18 in snakehead (Channa argus) during Aeromonas schubertii and Nocardia seriolae infections.

As a proinflammatory cytokine of the interleukin-1 (IL-1) family, IL-18 plays important roles in host protection against bacterial, viral, and fungal infection. We cloned the open reading frame of snakehead (Channa argus) IL-18 (shIL-18) and found that it contained 609 base pairs and encoded 202 amino acid residues. The shIL-18 included a conserved IL-1-like family signature and two potential IL-1ß-converting enzyme cutting sites; one was conserved in all analyzed IL-18s, but the other was unique to shIL-18. Unlike other IL-18s, shIL-18 also contained a predicted signal peptide. In this study, shIL-18 was constitutively expressed in all tested tissues, and its expression was induced by Aeromonas schubertii and Nocardia seriolae in the head kidney and spleen in vivo and by lipoteichoic acid, lipopolysaccharides, and polyinosinic-polycytidylic acid in head kidney leukocytes in vitro. Moreover, recombinant shIL-18 upregulated the expression of interferon-γ, IL-1ß, and tumor necrosis factor-α1 and -α2 and promoted the proliferation of leukocytes. Taken together, these results showed that IL-18 played crucial roles in host defense against bacterial infection in fish, as it does in mammals.

MEKK3 in hybrid snakehead (Channa maculate ♀ ×Channa argus ♂): Molecular characterization and immune response to infection with Nocardia seriolae and Aeromonas schubertii.

Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 3 (MEKK3) is a serine/threonine protein kinase that acts as a key regulator and is widely involved in various innate and acquired immune signaling pathways. In this study, we first cloned the complete open reading frame (ORF) of the MEKK3 gene (named CcMEKK3) in a hybrid snakehead (Channa maculate ♀ × Channa argus ♂). The full-length ORF of CcMEKK3 is 1851 bp, and encodes a putative protein of 616 amino acids containing a serine/threonine kinase catalytic (S-TKc) domain and a Phox and Bem1p (PB1) domain. A sequence alignment and phylogenetic tree analysis showed that CcMEKK3 is highly conserved relative to the MEKK3 proteins of other teleost species. CcMEKK3 was constitutively expressed in all the healthy hybrid snakehead tissues tested, with greatest expression in the immune tissues, such as the head kidney and spleen. The expression of CcMEKK3 was usually upregulated in the head kidney, spleen, and liver at different time points after infection with Nocardia seriolae or Aeromonas schubertii. Similarly, the dynamic expression levels of CcMEKK3 in head kidney leukocytes after stimulation revealed that CcMEKK3 was induced by LTA, LPS, and poly(I:C). In the subcellular localization analysis, CcMEKK3 was evenly distributed in the cytoplasm of HEK293T cells, and its overexpression significantly promoted the activities of NF-κB and AP-1. These results suggest that CcMEKK3 is involved in the immune defense against these two pathogens, and plays a crucial role in activating the NF-κB and MAPK signaling pathways.

Elucidation of the Interleukin 12 Production Mechanism during Intracellular Bacterial Infection in Amberjack, Seriola dumerili.

Intracellular bacterial infections affect all vertebrates. Cultured fish are particularly vulnerable because no effective protection measures have been established since such infections emerged approximately 50 years ago. As in other vertebrates, the induction of cell-mediated immunity (CMI) plays an important role in protecting fish against infection. However, details of the mechanism of CMI induction in fish have not been clarified. In the present study, we focused on the production of interleukin 12 (IL-12), an important factor in CMI induction in fish. Using several different approaches, we investigated IL-12 regulation in amberjack (Seriola dumerili), the species most vulnerable to intracellular bacterial disease. The results of promoter assays and transcription factor gene expression analyses showed that the expression of interferon regulatory factor-1 (IRF-1) and activator protein-1 (AP-1) is necessary for IL-12 production. Phagocytosis of living cells (LCs) of Nocardia seriolae bacteria induced IL-12 production in neutrophils, accompanied by IRF-1 and AP-1 gene expression. Bacteria in which the exported repetitive protein (Erp)-like gene was deleted (Δerp-L) could not establish intracellular parasitism or induce IRF-1 and AP-1 expression or IL-12 production, despite being phagocytosed by neutrophils. These data suggest that IL-12 production is regulated by (i) two transcription factors, IRF-1 and AP-1, (ii) phagocytosis of LCs by neutrophils, and (iii) one or more cell components of LCs. Our results enhance the understanding of the immune response to intracellular bacterial infections in vertebrates and could facilitate the discovery of new agents to prevent intracellular bacterial disease.

Cutaneous nocardiosis with discharging sinus clinically mimicking tuberculosis diagnosed by cytology.

Nocardiosis is primarily a pulmonary infection commonly seen in immunocompromised individuals. However, lymphocutaneous nocardiosis is observed in immunocompetent individuals often after trauma. The clinical and cytomorphological features of lymphocutaneous nocardiosis closely mimic the most common infections in India such as tuberculosis and mycetoma (very common cutaneous infection with discharging sinus). As it is crucial to differentiate nocardiosis from tuberculosis, to avoid unnecessary antitubercular treatment, special stains like modified Ziehl-Neelsen stain and Gram stain can be employed to differentiate the morphology of Nocardia from tuberculosis. Fine-needle cytology from these cutaneous lesions helps in yielding adequate material for rapid and accurate diagnosis of immediate specific antibiotic treatment. We report a rare case that presented with clinical diagnosis of tuberculosis but turned out to be nocardiosis on cytomorphology with simple and most feasible fine-needle aspiration method of tissue diagnosis and scrape cytology.

Dehydropropylpantothenamide isolated by a co-culture of Nocardia tenerifensis IFM 10554T in the presence of animal cells.

A new amide, named dehydropropylpantothenamide (1), was obtained by a co-culture of Nocardia tenerifensis IFM 10554T in the presence of the mouse macrophage-like cell line J774.1 in modified Czapek-Dox (mCD) medium. Compound 1 was synthesized from D-pantothenic acid calcium salt in 6 steps. The absolute configuration of natural compound 1 was determined by comparisons of the optical rotation and CD spectra of synthetic 1. In the present study, a new method for producing secondary metabolites was demonstrated using a "co-culture" in which the genus Nocardia was cultured in the presence of an animal cell line.

Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion.

Gastro-intestinal helminth infections trigger the release of interleukin-33 (IL-33), which induces type-2 helper T cells (Th2 cells) at the site of infection to produce IL-13, thereby contributing to host resistance in a T cell receptor (TCR)-independent manner. Here, we show that, as a prerequisite for IL-33-induced IL-13 secretion, Th2 cells required the expression of the epidermal growth factor receptor (EGFR) and of its ligand, amphiregulin, for the formation of a signaling complex between T1/ST2 (the IL-33R) and EGFR. This shared signaling complex allowed IL-33 to induce the EGFR-mediated activation of the MAP-kinase signaling pathway and consequently the expression of IL-13. Lack of EGFR expression on T cells abrogated IL-13 expression in infected tissues and impaired host resistance. EGFR expression on Th2 cells was TCR-signaling dependent, and therefore, our data reveal a mechanism by which antigen presentation controls the innate effector function of Th2 cells at the site of inflammation.

Synergism between genome sequencing, tandem mass spectrometry and bio-inspired synthesis reveals insights into nocardioazine B biogenesis.

Marine actinomycete-derived natural products continue to inspire chemical and biological investigations. Nocardioazines A and B (3 and 4), from Nocardiopsis sp. CMB-M0232, are structurally unique alkaloids featuring a 2,5-diketopiperazine (DKP) core functionalized with indole C3-prenyl as well as indole C3- and N-methyl groups. The logic of their assembly remains cryptic. Bioinformatics analyses of the Nocardiopsis sp. CMB-M0232 draft genome afforded the noz cluster, split across two regions of the genome, and encoding putative open reading frames with roles in nocardioazine biosynthesis, including cyclodipeptide synthase (CDPS), prenyltransferase, methyltransferase, and cytochrome P450 homologs. Heterologous expression of a twelve gene contig from the noz cluster in Streptomyces coelicolor resulted in accumulation of cyclo-l-Trp-l-Trp DKP (5). This experimentally connected the noz cluster to indole alkaloid natural product biosynthesis. Results from bioinformatics analyses of the noz pathway along with challenges in actinomycete genetics prompted us to use asymmetric synthesis and mass spectrometry to determine biosynthetic intermediates in the noz pathway. The structures of hypothesized biosynthetic intermediates 5 and 12-17 were firmly established through chemical synthesis. LC-MS and MS-MS comparison of these synthetic compounds with metabolites present in chemical extracts from Nocardiopsis sp. CMB-M0232 revealed which of these hypothesized intermediates were relevant in the nocardioazine biosynthetic pathway. This established the early and mid-stages of the biosynthetic pathway, demonstrating that Nocardiopsis performs indole C3-methylation prior to indole C3-normal prenylation and indole N1'-methylation in nocardioazine B assembly. These results highlight the utility of merging bioinformatics analyses, asymmetric synthetic approaches, and mass spectrometric metabolite profiling in probing natural product biosynthesis.

Nocardia otitidiscaviarum (GAM-5) induces parkinsonian-like alterations in mouse.

Parkinson's disease, a major neurodegenerative disorder in humans whose etiology is unknown, may be associated with some environmental factors. Nocardia otitidiscaviarum (GAM-5) isolated from a patient with an actinomycetoma produced signs similar to Parkinson's disease following iv injection into NMRI mice. NMRI mice were infected intravenously with a non-lethal dose of 5 x 10(6) colony forming units of N. otitidiscaviarum (GAM-5). Fourteen days after bacterial infection, most of the 60 mice injected exhibited parkinsonian features characterized by vertical head tremor, akinesia/bradykinesia, flexed posture and postural instability. There was a peak of nocardial growth in the brain during the first 24 h followed by a decrease, so that by 14 days nocardiae could no longer be cultured. At 24 h after infection, Gram staining showed nocardiae in neurons in the substantia nigra and occasionally in the brain parenchyma in the frontal and parietal cortex. At 21 days post-infection, tyrosine hydroxylase immunolabeling showed a 58% reduction of tyrosine hydroxylase in the substantia nigra, and a 35% reduction of tyrosine hydroxylase in the ventral tegmental region. Dopamine levels were reduced from 110 +/- 32.5 to 58 +/- 16.5 ng/mg protein (47.2% reduction) in brain from infected mice exhibiting impaired movements, whereas serotonin levels were unchanged (191 +/- 44 protein in control and 175 +/- 39 ng/mg protein in injected mice). At later times, intraneuronal inclusion bodies were observed in the substantia nigra. Our observations emphasize the need for further studies of the potential association between Parkinson's disease or parkinsonism-like disease and exposure to various nocardial species.

Neuroinvasive Nocardia asteroides GUH-2 induces apoptosis in the substantia nigra in vivo and dopaminergic cells in vitro.

Neurodegenerative diseases such as Parkinson's disease are increasingly prevalent in the aging population worldwide. The causes of these disorders are unknown, but many studies have suggested that the etiology is likely multifactorial and may involve exposure to something in the environment combined with the normal aging process. Nocardia asteroides are bacteria commonly found in the soil, and neuroinvasive strains of nocardiae have been described. N. asteroides strain GUH-2 invades the brains of experimentally infected animals and selectively affects dopaminergic neurons of the substantia nigra (SN), causing an L-DOPA-responsive movement disorder resembling parkinsonism. Furthermore, dopaminergic neurons undergo morphological changes characteristic of apoptosis following nocardial infection. Apoptosis has been implicated in dopaminergic neuronal dropout in Parkinson's patients as well as other parkinsonian models. Thus, in this study, in vivo and in vitro models were utilized to measure the ability of GUH-2 to induce the apoptotic death of dopaminergic cells. Following infection with GUH-2, dopaminergic apoptotic cells were identified in the SN of animals by in situ end labeling, which detects DNA fragmentation, combined with fluorescent immunolabeling of tyrosine hydroxylase-positive cells. In addition, apoptosis was observed in PC12 cell cultures incubated with GUH-2 by both in situ end labeling and the annexin V assay, which detects externalization of phosphatidylserine of the plasma membrane, indicating apoptotic death. Based on the results of these studies, it appears that experimental infection with N. asteroides provides a general model for studying apoptosis in parkinsonian disorders.

Monoamine changes in the brain of BALB/c mice following sub-lethal infection with Nocardia asteroides (GUH-2).

BALB/c mice injected intravenously with a single, sub-lethal dose of Nocardia asteroides GUH-2 develop several levodopa responsive movement disorders. These included headshake, stooped posture, bradykinesia, and hesitation to forward movement. The changes in monoamine levels in the brain of these mice were determined. There was a significant loss of dopamine with greatly increased dopamine turnover in the neostriatum 7 to 29 days after infection. These effects were specific for dopaminergic neurons since minimal changes were found in neostriatal norepinephrine and serotonin even though serotonin turnover was increased. Changes in monoamine metabolism were not limited to the neostriatum. There were reduced levels of serotonin and norepinephrine with increased serotonin turnover in the cerebellum. One year after infection, dopamine metabolism had returned to near normal levels, but many of the movement disorders persisted. Specific changes in neurochemistry did not always appear to correspond with these impairments. Nevertheless, these data are similar to those reported in MPTP treated BALB/c mice.

Reversible alterations in brain metabolites during therapy for disseminated nocardiosis using proton magnetic resonance spectroscopy.

We report reversible abnormalities in magnetic resonance spectra acquired from a patient with AIDS undergoing antibiotic and corticosteroid therapy for disseminated nocardiosis, a rare opportunistic infection of immunosuppressed patients which can cause cerebral abscess formation. There was no clinical, CT or MRI evidence of HIV-1 encephalitis. MR spectra were acquired before and after treatment using a two-dimensional chemical shift imaging technique (TR 1500ms, TE 130ms). Prior to treatment, a rise in the choline to creatine ratio and a reduction in the N-acetylaspartate to creatine ratio were observed in MR spectra localized to areas of the left anteromedial centrum semiovale that appeared normal on MR imaging. After 16 weeks, the patient had recovered with complete resolution of the cerebral abscesses on MRI. The MR spectral abnormalities also returned to normal. Two months later, the patient had a relapse with focal neurological signs and further abscesses were demonstrated on MRI of the brain. The patient subsequently died and histopathological and microbiological findings at autopsy confirmed the clinical picture of a recurrence of cerebral nocardiosis with no evidence of HIV-1 encephalitis. This case illustrates reversible MR-measurable metabolite changes in the brain of an HIV-seropositive patient without HIV-1 encephalitis who underwent treatment for cerebral nocardiosis.

Acquired resistance of Nocardia brasiliensis to clavulanic acid related to a change in beta-lactamase following therapy with amoxicillin-clavulanic acid.

Previous studies have demonstrated that Nocardia brasiliensis is susceptible to amoxicillin-clavulanic acid and that its beta-lactamases are inhibited in vitro by clavulanic acid. A cardiac transplant patient with disseminated infection caused by N. brasiliensis was treated with this drug combination with good response, but relapsed while still on therapy. The relapse isolate was found to be identical to the initial isolate by using genomic DNA restriction fragment patterns obtained by pulsed field gel electrophoresis, but it was resistant to amoxicillin-clavulanic acid. On isoelectric focusing, the beta-lactamase from the relapse isolate exhibited a shift in the isoelectric point (pI) of its major band from 5.10 to 5.04 compared with the enzyme from the pretreatment isolate. As determined by using values of the amount of beta-lactamase inhibitor necessary to give 50 +/- 5% inhibition of beta-lactamase-mediated hydrolysis of 50 microM nitrocefin, the beta-lactamase of the relapse isolate was also 200-fold more resistant than the enzyme from the pretreatment isolate to clavulanic acid and was more resistant to sulbactam, tazobactam, cloxacillin, and imipenem. The beta-lactamase of the relapse isolate exhibited a 10-fold decrease in hydrolytic activity for cephaloridine and other hydrolyzable cephalosporins compared with that for nitrocefin. Acquired resistance to amoxicillin-clavulanic acid in this isolate of N. brasiliensis appears to have resulted from a mutational change affecting the inhibitor and active site(s) in the beta-lactamase.

[Nocardial brain abscess: case report].

A 25-year-old man, who was slightly immunosuppressed, presented headache and right motor weakness due to multiple brain abscesses disseminated from lung abscess. They were diagnosed, by bacteriological examination, as nocardial brain abscesses (nocardia asteroides) 4 weeks after the first operation. In spite of delay in the diagnosis, he was relieved by operations (three times) and chemotherapy including high doses of Sulfamethoxazole-Trimethoprim. He was eventually discharged. We stress the necessity of early diagnosis and the efficacy of Sulfamethoxazole-Trimethoprim for nocardial brain abscess.

Treatment of Nocardia asteroides infection with trimethoprim-sulfamethoxazole.

Although sulfonamide therapy has reduced the case fatality rate in infection due to Nocardia asteroides from nearly 100% to 25% to 45% there remains a need for a still more effective chemotherapeutic regimen. We describe three cases of serious infection due to N asteroides treated successfully with trimethoprim-sulfamethoxazole (TMP-SMX) and review an additional 15 cases from the world literature. Trimethoprim and sulfamethoxazole exhibit a synergistic interaction in vitro against N asteroides, and the agent reaches antibacterial concentrations in blood, lung, and the central nervous system. Clinical results have been satisfactory in the limited number of patients treated to date.