The Signaling Pathway of Caenorhabditis elegans Mediates Chemotaxis Response to the Attractant 2-Heptanone in a Trojan Horse-like Pathogenesis.

The nematode Caenorhabditis elegans exhibits behavioral responses to a wide range of odorants associated with food and pathogens. A previous study described a Trojan Horse-like strategy of pathogenesis whereby the bacterium Bacillus nematocida B16 emits the volatile organic compound 2-heptanone to trap C. elegans for successful infection. Here, we further explored the receptor for 2-heptanone as well as the pathway involved in signal transduction in C. elegans Our experiments showed that 2-heptanone sensing depended on the function of AWC neurons and a GPCR encoded by str-2 Consistent with the above observation, the HEK293 cells expressing STR-2 on their surfaces showed a transient elevation in intracellular Ca2+ levels after 2-heptanone applications. After combining the assays of RNA interference and gene mutants, we also identified the Gα subunits and their downstream components in the olfactory signal cascade that are necessary for responding to 2-heptanone, including Gα subunits of egl-30 and gpa-3, phospholipase C of plc-1and egl-8, and the calcium channel of cmk-1 and cal-1. Our work demonstrates for the first time that an integrated signaling pathway for 2-heptanone response in C. elegans involves recognition by GPCR STR-2, activation by Gα subunits of egl-30/gpa-3 and transfer to the PLC pathway, indicating that a potentially novel olfactory pathway exists in AWC neurons. Meanwhile, since 2-heptanone, a metabolite from the pathogenic bacterium B. nematocida B16, can be sensed by C. elegans and thus strongly attract its host, our current work also suggested coevolution between the pathogenic microorganism and the chemosensory system in C. elegans.

Cytokines and olfactory bulb microglia in response to bacterial challenge in the compromised primary olfactory pathway.

BACKGROUND: The primary olfactory pathway is a potential route through which microorganisms from the periphery could potentially access the central nervous system. Our previous studies demonstrated that if the olfactory epithelium was damaged, bacteria administered into the nasal cavity induced nitric oxide production in olfactory ensheathing cells. This study investigates the cytokine profile of olfactory tissues as a consequence of bacterial challenge and establishes whether or not the bacteria are able to reach the olfactory bulb in the central nervous system. METHODS: The olfactory epithelium of C57BL/6 mice was damaged by unilateral Triton X-100 nasal washing, and Staphylococcus aureus was administered ipsilaterally 4 days later. Olfactory mucosa and bulb were harvested 6 h, 24 h and 5 days after inoculation and their cytokine profile compared to control tissues. The fate of S. aureus and the response of bulbar microglia were examined using fluorescence microscopy and transmission electron microscopy. RESULTS: In the olfactory mucosa, administered S. aureus was present in supporting cells of the olfactory epithelium, and macrophages and olfactory nerve bundles in the lamina propria. Fluorescein isothiocyanate-conjugated S. aureus was observed within the olfactory mucosa and bulb 6 h after inoculation, but remained restricted to the peripheral layers up to 5 days later. At the 24-h time point, the level of interleukin-6 (IL-6) and tumour necrosis factor-α in the compromised olfactory tissues challenged with bacteria (12,466 ± 956 pg/ml and 552 ± 193 pg/ml, respectively) was significantly higher than that in compromised olfactory tissues alone (6,092 ± 1,403 pg/ml and 80 ± 2 pg/ml, respectively). Immunohistochemistry confirmed that IL-6 was present in several cell types including olfactory ensheathing cells and mitral cells of the olfactory bulb. Concurrently, there was a 4.4-, 4.5- and 2.8-fold increase in the density of iNOS-expressing cells in the olfactory mucosa, olfactory nerve and glomerular layers combined, and granule layer of the olfactory bulb, respectively. CONCLUSIONS: Bacteria are able to penetrate the immunological defence of the compromised olfactory mucosa and infiltrate the olfactory bulb within 6 h even though a proinflammatory profile is mounted. Activated microglia may have a role in restricting bacteria to the outer layers of the olfactory bulb.

A study of the lesions induced in Seriola dumerili by intradermal or intraperitoneal injection of Streptococcus dysgalactiae.

Fish of the species Seriola dumerili were infected experimentally with the pathogen Streptococcus dysgalactiae. Intradermal (ID) injection of S. dysgalactiae resulted in moderate mortality regardless of the dose of bacteria injected, whereas intraperitoneal (IP) injection caused greatest mortality in the group of fish receiving the highest dose of bacteria (10(9) colony forming units/ml). On necropsy examination of affected fish, the most striking change was microabscessation and/or granulomatous inflammation of the heart, caudal peduncle, pectoral and/or dorsal fin and olfactory region. The lesions in the atrial myocardium and arterial cone consisted of severe arterial thrombosis, granulomatous valvular endocarditis and epicarditis. S. dysgalactiae was cultured from these lesions and S. dysgalactiae antigen was demonstrated by immunohistochemistry within these tissues. The mortality in these fish is therefore considered to reflect bacterial septicaemia and systemic granulomatous inflammatory disease.

Pneumococcal carriage results in ganglioside-mediated olfactory tissue infection.

Streptococcus pneumoniae cause considerable morbidity and mortality, with persistent neurological sequelae, particularly in young children and the elderly. It is widely assumed that carriage occurs through direct mucosal colonization from the environment whereas meningitis results from invasion from the blood. However, the results of published studies can be interpreted that pneumococci may enter the brain directly from the nasal cavity by axonal transport through olfactory nerves. This hypothesis is based on findings that (i) teichoic acid of the pneumococcal cell wall interact with gangliosides (GLS), (ii) the interaction of GLS with cholera toxin leads to axonal transport through the olfactory nerves into the brain, and (iii) viruses enter the brain through axonal transport into olfactory nerves. After nasal inoculation, we observe high numbers of pneumococci in nasal washes and the olfactory nerves and epithelium. Significant numbers of pneumococci also infected the olfactory bulbs, brain, and the trigeminal ganglia. The absence of bacteremia in this model makes it unlikely that the bacteria entered the brain from the blood stream. Recovery of colony-forming units from the brain, lungs, olfactory nerves, and epithelium and nasal washes was inhibited by incubating pneumococci with GLS before nasal inoculation. These findings, confirmed by PCR and immunohistochemistry, support a GLS-mediated process of infection and are consistent with pneumococci reaching the brain through retrograde axonal transport.

Role of envelope glycoproteins gI, gp63 and gIII in the invasion and spread of Aujeszky's disease virus in the olfactory nervous pathway of the pig.

One-week-old pigs were infected intranasally with the Ka strain of Aujeszky's disease virus (ADV) or with mutants that were lacking the non-essential envelope glycoproteins gI, gp63 or gIII. The invasion and spread of these strains in the olfactory nervous pathway were examined by assessing virus levels and by localizing viral antigens in the olfactory mucosa representing the first neuronal level, in the olfactory bulb representing the second neuronal level and in the lateral olfactory gyrus, the rostral perforated substance and the piriform lobe, all representing the third neuronal level. The Ka parental strain invaded and spread up to the third neuronal level. The extent of invasion and spread of the gIII- mutant were similar to those of the parental strain. The gp63- mutant replicated normally in the olfactory mucosa, but its spread to all the other levels was limited as compared with that of the parental strain. The gI- mutant showed a defect in infection at all neuronal levels. These results indicate that, of the non-essential envelope glycoproteins, gI plays the major role in neural invasion and spread of ADV in its natural host. The pattern of invasion and spread of these mutants in the olfactory pathway of pigs was similar to that previously observed in the trigeminal pathway. The type of nervous pathway therefore appears not to influence the neuropathogenesis of ADV or mutants deleted in non-essential envelope glycoproteins in the pig.

Olfactory pathways in three patients with cryptococcal meningitis and acquired immune deficiency syndrome.

The olfactory mucosa, bulbs and tracts were examined for the presence of Cryptococcus neoformans in 3 patients with the acquired immune deficiency syndrome (AIDS) and cryptococcal meningitis. Two of them had antibodies against HIV-1 and one had positive serology for HIV-2. Cryptococci were seen in the subarachnoid space around olfactory tracts and bulbs and in the submucosal olfactory nerve fascicles. In one case, olfactory nerve fascicles from the lamina propria were also affected. Olfactory epithelium and respiratory mucosa were not involved. We suggest that Cryptococcus reached the olfactory nerve fascicles through the olfactory pathways for cerebrospinal fluid drainage which might serve as a source of latent cryptococcal infection.

The CVS strain of rabies virus as transneuronal tracer in the olfactory system of mice.

The sequential distribution of transneuronally infected neurons was studied in the olfactory pathway of mice after unilateral inoculation of the challenge virus standard (CVS) strain in the nasal cavity. A first cycle of viral multiplication was observed in a subpopulation of receptor cells scattered in the main olfactory epithelium and in the septal organ. No viral spread from cell body to cell body was reported even in later stages of infection. The second round of viral replication which took place in the ipsilateral main olfactory bulb at 2 and 2.5 days post-inoculation (p.i.), involved second order neurons and periglomerular cells, known to be directly connected with the axon terminals of receptor cells. Also reported as a result of a second cycle of viral replication, was surprisingly the spread of CVS at 2 and 2.5 days p.i. in bulbar interneurons located in the internal plexiform layer and in the superficial granule cell layer, as well as that of 2 ipsilateral cerebral nuclei, the anterior olfactory nucleus and the horizontal limb of the diagonal band. From day 3, a rapid spread of CVS was suggested by detection of virus in all ipsilateral direct terminal regions of the second order neurons and in most tertiary olfactory projections. The locus coeruleus, a noradrenergic nucleus which sends direct afferents to the olfactory bulb, never appeared immunoreactive. In spite of a certain inability of CVS to infect some neuron types, the virus appears relevant to provide new information regarding the complex network of olfactory-related neurons into the CNS.

Viral infection and dissemination through the olfactory pathway and the limbic system by Theiler's virus.

Theiler's murine encephalomyelitis virus (TMEV) infection of mice can produce a biphasic disease of the central nervous system (CNS). Most susceptible strains of mice survive the acute infection and develop a chronic demyelinating disease. In this report, we analyzed the routes of spread of TMEV within the CNS of nude mice and target sites eventually infected in the CNS. Compared to the immunocompetent mouse, in which an antiviral immune response is mounted but virus persists, the nude mouse develops a severe encephalomyelitis due to the lack of functional T lymphocytes and provides a useful model for the study of viral dissemination. We demonstrated, by immunohistochemistry, the presence of viral antigen in defined regions of the CNS, corresponding to various structures of the limbic system. In addition, we found a different time course for viral spread using two different sites of intracerebral inoculation, ie, via the olfactory bulb or the cortex. Limbic structures were rapidly infected following olfactory bulb infection and then showed a decrease in viral load, presumably due to loss of target neurons. Using either route of infection, the virus was able to disseminate to similar regions. These results indicate that limbic structures and their connections are very important for the spread of TMEV in the brain. In the spinal cord, not only neuronal but hematogenous pathways were suspected to be involved in the dissemination of Theiler's virus.

Mouse hepatitis virus and herpes simplex virus move along different CNS pathways.

The spread of mouse hepatitis virus, strain JHM and herpes simplex virus type 1 in the central nervous system after inoculation into the nares and main olfactory bulb has been examined. The results show that each virus infects a subset of the possible connections of the olfactory bulb and that the subset infected by each virus is different. Thus, both viruses will be useful for studying the neuroanatomic connections of the olfactory bulb, and possibly for functional analyses as well.

Immunohistological demonstration of spread of Aujeszky's disease virus via the olfactory pathway in HPCD pigs.

The spread of Aujeszky's disease virus (ADV) from nasal mucosa via the olfactory pathway was studied in HPCD pigs. ADV antigen was detected in the epithelial cells, nasal gland cells, olfactory nerve cells and peripheral nerve fibres in the nasal cavity and in neuroglial cells in the olfactory bulb. Results indicate that the olfactory pathway is one of the most important neuronal pathways of ADV infection in pigs.

Spread of the CVS strain of rabies virus and of the avirulent mutant AvO1 along the olfactory pathways of the mouse after intranasal inoculation.

After intranasal instillation in the mouse, rabies virus (CVS strain) selectively infected olfactory receptor cells. In the main olfactory bulb (MOB), infection was observed in periglomerular, tufted, and mitral cells and in interneurons located in the internal plexiform layer. Beyond the MOB, CVS spread into the brain along the olfactory pathways. This infection is specific to chains of functionally related neurons but at the death of the animal some nuclei remain uninfected. CVS also penetrated the trigeminal system. The avirulent mutant AvO1, carrying a mutation in position 333 of the glycoprotein, infected the olfactory epithelium and the trigeminal nerve as efficiently as CVS. During the second cycle of infection, the mutant was able to infect efficiently periglomerular cells in the MOB and neurons of the horizontal limb of the diagonal band, which indicates that maturation of infective particles is not affected in primarily infected neuronal cells. On the other hand, other neuronal cells permissive for CVS, such as mitral cells or the anterior olfactory nucleus, are completely free of infection with the mutant, indicating that restriction is related to the ability of AvO1 to penetrate several categories of neurons. From these observations, we concluded that CVS should be able to bind several different receptors to penetrate neurons, while the mutant would be unable to recognize some of them.

Venezuelan equine encephalitis virus propagation in the olfactory tract of normal and immunized mice.

The role of virus spread in the induction of damage to the central nervous system (CNS) of mice infected with Venezuelan equine encephalitis virus (VEEV) via the respiratory route was studied. The virus concentration in various organs and in the blood, the sensitivity to different doses of virus, and ultrastructural lesions in various tissues were examined. It is concluded that VEEV can enter the CNS of nonimmunized mice both by vascular and by olfactory pathways, whereas in immunized mice the olfactory pathway is the main route.

Axonal transport of Borna disease virus along olfactory pathways in spontaneously and experimentally infected rats.

In this study it has been shown that infection of mother rats by Borna disease virus (BDV) from infected newborns led to a fatal disease. This differed both in clinical symptoms and in histological alterations from the form of the disease which occurred after intracerebral (i.c.) infection. Both parameters were, however, similar to those seen after experimental intranasal (i.n.) infection of adult rats. Detailed immunohistological studies showed clearly that after experimental i.n. infection, the infecting virus migrates intraaxonally from the neuroreceptors in the olfactory epithelium into the brain. It is therefore suggested that i.n. transmission is an important route of natural BDV infection.

Olfactory neural pathway in mouse hepatitis virus nasoencephalitis.

The mechanism of brain infection with mouse hepatitis virus-JHM was studied in BALB/cByJ mice following intranasal inoculation, and found to be a consequence of direct viral spread along olfactory nerves into olfactory bulbs of the brain. Infection was followed sequentially from nose to brain, using microscopy, immunohistochemistry and virus quantification. Lesions, antigen and virus were observed in the olfactory bulb and anterior brain as early as 2 days and posterior brain by 4 days after inoculation. Viral antigen extended through nasal mucosa into submucosa, then coursed along the olfactory nerve perineurium and fibers, through the cribriform plate into the olfactory bulbs. On days 4 and 7, viral antigen was found in the antero-ventral brain, along ventral meninges, olfactory tracts and anterior ramifications of the lateral ventricles. Virus was cleared from nose by 10 days and anterior brain by 20 days, but persisted in posterior brain for 20 days after inoculation. Mice also developed disseminated infection, with viremia and hepatitis. Infection of brain did not correlate with presence of viremia. In contrast to intranasally inoculated mice, orally-inoculated mice did not develop encephalitis, despite evidence of disseminated infection.

Herpes simplex encephalitis. Immunohistological demonstration of spread of virus via olfactory and trigeminal pathways after infection of facial skin in mice.

An immunohistological study of viral antigen (VA) in the brain was carried out in mice which had been infected with herpes simplex type 1 virus (HSV) in the skin of the face. In 77% of the mice with VA in the brain the olfactory system as well as the trigeminal system/brainstem was affected. The remaining 23% had VA in the trigeminal system/brainstem only. Eye swab cultures yielded HSV from all mice with VA in the olfactory system. The ease of access of virus infecting the face to the olfactory system shown in this model may have implications for human infections.

The distribution of herpes simplex type 1 antigen in mouse central nervous system after different routes of inoculation.

Herpes simplex type 1 virus was inoculated into 3-week-old mice via four different routes; intracerebral, intravenous, intranasal and directly into the sciatic nerve. Virus antigen-containing cells in the central nervous system were identified by both an immunofluorescence and immunoperoxidase method. The portal of entry of virus into the CNS appeared to be the major determinant of distribution of virus antigen. Direct haematogenous seeding of virus into the CNS was not proven. It seems probable that infection was first established in sensory ganglia. Within the CNS, regions of high virus antigen concentration paralleled high cell density suggesting cell to cell spread. Consistent involvement of certain neuron groups may be due to their selective vulnerability. These animal experiments provide some explanation for the patterns of CNS herpetic infection observed in man.

Subarachnoid space of the CNS, nasal mucosa, and lymphatic system.

We have briefly reviewed the literature pertaining to the movement of tracer molecules and infectious organisms within the olfactory nerve. There is a body of evidence indicating that tracers placed in the CSF will quickly move via the olfactory nerve to the nasal mucosa and then to the cervical lymph nodes. Organic and inorganic tracer materials and organisms as diverse as viruses, a bacillus, and an amoeba, when placed in the nasal cavity, have been shown to move from the nasal mucosa via the olfactory nerve to the olfactory bulb and the CSF. We think that a portion of the data on tracer movement is due to incorporation of tracer materials and organisms into the axoplasm of the olfactory neurons with subsequent anterograde or retrograde axoplasmic transport. However, some of the movement of tracers may occur within the olfactory perineural space. This space may be continuous with a subarachnoid extension that surrounds the olfactory nerve as it penetrates the cribriform plate. To our knowledge, no one has yet followed the perineural space to determine if it is continuous from olfactory receptor to olfactory bulb. The consideration of this space and its role is the main reason for this review.