Change in the protective activity of the ribosomal fractions of Salmonella typhimurium in mutations in the rfa region and the transduction replacement of the rfb region]

It was shown that mutation in the rfa region causing disturbances in the structure of the basal part of the polysaccharide of the cell wall or the absence of O-specific side chains led to the loss of protective activity of the ribosomal fractions isolated from the cells of the murine typhoid salmonella by sedimentation with dihydrostreptomycine sulphate. Ribosomal fractions isolated from the murine typhoid salmonella transductants with the replaced rfb region failed to protect the animals from the infection with the virulent. S. typhimurium, S. enteritidis cultures. The virulence of the mutants and transductants was also changed in comparison with the initial strains.     

The primary effect of the Ity locus is on the rate of growth of Salmonella typhimurium that are relatively protected from killing

The Ity locus affects the net increase in numbers of Salmonella typhimurium in the liver and spleen of infected mice. There has been controversy, however, about whether the effects of this locus are due to differential killing of S. typhimurium or differential growth rates of S. typhimurium in mice. Our studies using S. typhimurium aroA mutants, which do not grow in vivo, demonstrate that growth of the infecting salmonella is necessary for the observation of the Ity phenotype. To examine the effects of the Ity locus on the growth and killing of fully virulent salmonella, we infected Ity-congenic mice i.v. with stationary phase S. typhimurium containing a single copy of the plasmid pHSG422. This plasmid exhibits defective replication at body temperature and is diluted out during salmonella growth in vivo. Thus, the frequency of plasmid-containing salmonella recovered from mice provides a measure of salmonella cell divisions in vivo. Inasmuch as the numbers of plasmid-containing salmonella are only slightly affected by bacterial division, any decline in the numbers of plasmid-containing salmonella is an unbiased measure of killing. By infecting mice with these plasmid-containing salmonella we observed that: 1) during the first four h post infection (during blood clearance of injected salmonella) there is about 3-fold more killing of salmonella in Ityr mice than in Itys mice; 2) from 4 to 44 h postinfection (after blood clearance is completed) there is little if any additional killing in either Itys or Ityr mice; and 3) during the first 48 h postinfection there is about 18-fold more growth of salmonella in Itys mice than in Ityr mice. Thus, the major effect of the Ity locus on resistance to salmonella, is the regulation of growth within a "safe" (relatively nonbactericidal) site in the liver and spleen.     

Selection of Salmonella enterica serovar Typhi genes involved during interaction with human macrophages by screening of a transposon mutant library

The human-adapted Salmonella enterica serovar Typhi (S. Typhi) causes a systemic infection known as typhoid fever. This disease relies on the ability of the bacterium to survive within macrophages. In order to identify genes involved during interaction with macrophages, a pool of approximately 10(5) transposon mutants of S. Typhi was subjected to three serial passages of 24 hours through human macrophages. Mutants recovered from infected macrophages (output) were compared to the initial pool (input) and those significantly underrepresented resulted in the identification of 130 genes encoding for cell membrane components, fimbriae, flagella, regulatory processes, pathogenesis, and many genes of unknown function. Defined deletions in 28 genes or gene clusters were created and mutants were evaluated in competitive and individual infection assays for uptake and intracellular survival during interaction with human macrophages. Overall, 26 mutants had defects in the competitive assay and 14 mutants had defects in the individual assay. Twelve mutants had defects in both assays, including acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, SPI-4, STY1867-68, and STY2346. The complementation of several mutants by expression of plasmid-borne wild-type genes or gene clusters reversed defects, confirming that the phenotypic impairments within macrophages were gene-specific. In this study, 35 novel phenotypes of either uptake or intracellular survival in macrophages were associated with Salmonella genes. Moreover, these results reveal several genes encoding molecular mechanisms not previously known to be involved in systemic infection by human-adapted typhoidal Salmonella that will need to be elucidated.     

Inhibition of the Metabolism of Streptococci and Salmonella by Specific Antisera

Streptococcal and salmonella antisera inhibited carbohydrate metabolism for groups A, B, C, and D streptococci and group E salmonella, as measured by the formation of [(14)C]dioxide from [(14)C]glucose metabolism. For salmonella, the inhibition was type specific since group E salmonella were inhibited only by salmonella E antisera and not by anti-salmonella A or C(1). For streptococci, quantitative differences were demonstrated, but major cross-reactivity was observed. At high concentrations, the antisera were bactericidal; at more dilute concentrations, for both salmonella and streptococci, carbohydrate metabolism was suppressed, but subculture on chocolate agar showed abundant growth. Cross-reacting antibodies could be absorbed by incubation with either antigen, e.g., streptococcal antisera versus heat-killed salmonella. The results suggest that the radiometric technique can be more sensitive than either capillary flocculation or visual detection of bacterial growth for detecting the inhibition of streptococci and salmonella by specific antibodies. The use of specific antisera may prove useful for bacterial species identification in an automated system for detection of bacterial growth.     

Generation and characterization of a PhoP homologue mutant of Neisseria meningitidis

Two-component regulatory systems are important regulators of virulence genes in a number of bacteria. Genes encoding a two-component regulator system, with homology to the phoP/phoQ system in salmonella, were identified in the meningococcal genome. Allele replacement was used to generate a meningococcal knock-out mutant of the regulator component of this system, and its phenotype was examined. The mutant displayed many differences in protein profiles compared with wild type, consistent with it being a gene-regulatory mutation. Many of the growth characteristics of the mutant were similar to those of phoP mutants of salmonella: it was unable to grow at low concentrations of magnesium and was sensitive to defensins and other environmental stresses. Magnesium-regulated differences in protein expression were abrogated in the mutant, indicating that the meningococcal PhoP/PhoQ system may, as in salmonella, respond to changes in environmental magnesium levels. These results are consistent with the PhoP homologue playing a similar role in the meningococcus to PhoP in salmonella and suggest that it may similarly be involved in the regulation of virulence genes in response to environmental stimuli in the meningococcus. In support of this conclusion, we found the mutant grew was unable to grow in mouse serum and was attenuated in its ability to traverse through a layer of human epithelial cells. Identification of those genes regulated by the meningococcal PhoP may provide a route towards the identification of virulence genes in the meningococcus.     

Salmonella-induced macrophage death: multiple mechanisms, different outcomes

The facultative intracellular pathogen Salmonella enterica triggers programmed cell death in macrophages. The close examination of this phenomenon has revealed an unusually complex picture involving diverse mechanisms that lead to different types of programmed cell death. It appears that the outcome of the interaction of salmonella with macrophages depends on the relative contribution of two type III protein secretion systems, in conjunction with the stimulation of innate immunity outputs through conserved determinants collectively known as 'pathogen-associated molecular patterns' (PAMPs). These interactions result in a breakdown of the balance between survival and pro-apoptotic cellular pathways, which eventually leads to macrophage cell death. The relative significance for the infection process of the different types of macrophage cell death triggered by salmonella remains to be established.     

Salmonella-typhimurium-specific difference in rate of intracellular killing by resident peritoneal macrophages from salmonella-resistant CBA and salmonella-susceptible C57BL/10 mice

The aim of the present study was to determine whether the difference between the rate of intracellular killing of Salmonella typhimurium by macrophages of salmonella-resistant CBA and salmonella-susceptible C57BL/10 mice also holds for other salmonellae and other bacteria species. After in vivo phagocytosis, the initial rate of in vitro intracellular killing of S. typhimurium phagetype 505, S. typhimurium phagetype 510, and S. typhimurium M206 by macrophages of CBA mice amounted always to approximately 1.7 times the value found for macrophages of C57BL/10 mice (p less than 0.001), indicating that the difference in killing efficiency between CBA and C57BL/10 macrophages holds for various strains of S. typhimurium. However, some other salmonella species, i.e., S. dublin and S. heidelberg, as well as E. coli 054 and 02K1+, Listeria monocytogenes EGD and L347, and Staphylococcus aureus were killed equally efficiently by macrophages of both mouse strains. These findings indicate that the difference between the rates of intracellular killing by macrophages of salmonella-resistant CBA and salmonella-susceptible C57BL/10 does not hold for several other bacteria species and thus might be specific for S. typhimurium. Subsequent experiments showed that the in vivo proliferation of S. typhimurium 510 in the first 2 days after i.v. injection was 2.0-fold to 3.0-fold higher in the spleens and livers of C57BL/10 mice than in those of CBA mice, whereas the in vivo proliferation of S. dublin and S. heidelberg was between 1.0-fold to 1.4-fold higher in the C57BL/10 mice. These findings suggest that the differences between the rate of in vitro intracellular killing of salmonella by CBA and C57BL/10 macrophages are reflected in differences in the rate of in vivo proliferation of these microorganisms in CBA and C57BL/10 mice. To gain insight into the involvement of the oxidative metabolism of CBA and C57BL/10 macrophages in the difference in the rate of intracellular killing of S. typhimurium, the O2 consumption and H2O2 release by resident peritoneal macrophages was determined. The amplitudes of the respiratory burst and the release of H2O2 was identical in macrophages of the two mouse strains after triggering by either preopsonized heat-killed S. typhimurium or phorbol myristic acetate. These findings indicate that the mouse species-associated difference in the intracellular killing of S. typhimurium is not caused by a difference in the oxidative metabolism of CBA and C57BL/10 macrophages.     

Effect of Chlortetracycline Feeding on the Salmonella Reservoir in Chickens

An experimental model was developed to study the influence of antibiotic feeding on the salmonella reservoir in chickens. This model had the features of being very sensitive, allowing for the recovery of very low numbers of infecting salmonella, and, as was the case in the first experiment, closely simulated natural conditions in permitting the spread of salmonella from non-medicated carrier birds to their penmates. The data from both experiments demonstrate clearly that feeding a sub-therapeutic level of chlortetracycline did not increase the numbers of salmonella being shed into the environment. In fact, the salmonella pool was significantly reduced in chickens fed chlortetracycline at 200 g/ton. In the first experiment, conducted under more natural conditions, not only did the continuous use of chlortetracycline markedly reduce the spread of salmonella to a non-inoculated population of broiler chickens but it failed to cause the emergence of chlortetracycline-resistant salmonella. In the second experiment, where medicated feed was administered 6 days before the birds were individually infected with large numbers of salmonella, resistant organisms did appear; however these conditions did not lead to increased numbers of salmonella being shed.     

Isolation and biochemical characterization of partially transformation-defective mutants of avian myelocytomatosis virus strain MC29: localization of the mutation to the myc domain of the 110,000-dalton gag-myc polyprotein

Recently, we isolated three mutants of MC29 virus which, although able to transform fibroblasts with the same efficiency as wild-type MC29, were 100-fold less efficient at transforming macrophages. In this study we found that MC29-transformed quail producer cell line Q10 was able to generate these partially transformation defective mutants at a high frequency. Using tryptic peptide mapping, we determined that the smaller gag-myc polyproteins encoded by the transformation-defective viruses had lost myc-specific tryptic peptides. This suggested that the mutations which resulted in the transformation-defective viruses being inefficient at transforming macrophages were located in the v-myc sequence and thus directly implicated v-myc and the gag-myc polyprotein in transformation by MC29.     

Mouse chromosome 1 Ity locus regulates microbicidal activity of isolated peritoneal macrophages against a diverse group of intracellular and extracellular bacteria

The genotype of a mouse influences whether or not it will survive infection with the agent of murine typhoid, Salmonella typhimurium. The best-characterized murine salmonella response gene is a Chromosome 1 locus designated Ity. Inbred strains of mice that express the Itys allele are unable to contain the net growth of Salmonella typhimurium within their spleens and livers, and usually die early in the infection. By contrast, mice homozygous or heterozygous for the Ityr allele are able to control the net multiplication of Salmonella typhimurium within these organs. The Ity gene also appears to regulate the extent of replication within murine reticuloendothelial cell tissues of the obligate intracellular parasite Leishmania donovani, as well as the facultative intracellular bacteria Mycobacterium bovis and Mycobacterium lepraemurium. Previous studies from our laboratory strongly suggested that Ityr mice are more resistant to S. typhimurium infection than are Itys mice, because resident Ityr macrophages kill salmonellae more efficiently than do Itys macrophages. In this study, we used an in vitro macrophage assay to assess the specificity of the enhanced killing capacity of Ityr macrophages. We found that Ityr macrophages were better able than Itys macrophages to kill both intracellular bacteria (Salmonella typhi) and extracellular bacteria (Escherichia coli, Staphylococcus aureus, Corynebacterium diphtheriae). Thus, the diversity of organisms affected by Ity expression suggests that the product of this gene may play a key regulatory role in the initial interaction of mice with a variety of microbial agents.     

Incidence of the H2 group of plasmids in chloramphenicol-sensitive salmonella isolated in 1974 from clinical sources in Ontario.

Chloramphenicol resistance in salmonella obtained from clinical sources in Ontario was previously found to be often mediated by R plasmids of the H2 incompatibility group. In the present study 40 salmonella strains resistant to one or more of kanamycin, streptomycin, sulfonamides, or tetracycline but sensitive to chloramphenicol, were investigated to determine if they contained R plasmids. Self-transmissible plasmids were isolated from 17 of the strains, and 7 of those showed the bacteriophage inhibition and thermosensitive mechanism of transfer characteristic of H2 plasmids. Entry exclusion and incompatibility experiments confiremd their classification. The results demonstrate that in this population of salmonella, R plasmids of the H2 group are prevalent. Experiments with plasmid-specific phages indicate that the plasmids of this sample, which are not in the H2 group, do not belong to any of the F, I, N, P, or W incompatibility groups.     

沙门菌CWDMs苹果酸脱氢酶的检测

目的：了解沙门菌细菌壁缺陷突变株（CWDMs）的生物氧化及遗传特点和探讨细菌壁缺陷变异的性质与机制。方法：采用PAGE电泳法和分光光度法检测伤寒沙门菌和甲型副伤寒沙门菌及其CWDMs和伤寒沙门菌粗糙型和苹果酸脱氢酶（MDH）同工酶的活性与类型。结果：伤寒沙门菌和甲型副伤寒沙门菌的细菌型和伤寒沙门菌粗糙型经PAGE电泳可见一条MDH同工酶带,CWDMs电泳后可见两条MDH同Ⅰ酶带,在CWDMs的MDH中有一条泳动速率与细菌型及粗糙型的相同,另一条则较快。分光光度法检测证实。细菌型与粗糙型的MDH活性相似,CWDMs的MDH活性则明显较低。结论：CWDMs保留了与亲代细菌型一致的MDH和形成了一种新的MDH,并且其MDH的活性已显著降低,此特性可能与CWDMs生物氧化特性的改变有关。     

Yersinia has a tropism for B and T cell zones of lymph nodes that is independent of the type III secretion system

Pathogenic Yersinia have a pronounced tropism for lymphatic tissues and harbor a virulence plasmid that encodes a type III secretion system, pTTSS, that transports Yops into host cells. Yops are critical virulence factors that prevent phagocytosis by macrophages and neutrophils and Yersinia mutants lacking one or more Yops are defective for survival in lymphatic tissues, liver, and gastrointestinal tract. However, here we demonstrate that Y. pseudotuberculosis (Yptb) mutants lacking the pTTSS survived as well as or better than wild-type (WT) Yptb in the mesenteric lymph nodes (MLN). Infection with pTTSS mutants caused lymphadenitis with little necrosis, whereas infection with WT Yptb provoked lymphadenitis with multiple necrotic suppurative foci. Gentamicin protection assays and microscopic examination of the MLN revealed that pTTSS mutants resided extracellularly adjacent to B and T lymphocytes in the cortex and paracortex. WT Yptb was found extracellularly adjacent to neutrophils and macrophages in necrotic areas and adjacent to B and T lymphocytes in less-inflamed areas. To determine whether lymphocytes protected pTTSS mutants from phagocytic cells, Rag1(-/-) mice were infected with pTTSS mutants or WT Yptb. pTTSS mutants but not WT, were impaired for survival in MLN of Rag1(-/-) mice, suggesting that lymphocyte-rich regions constitute a protective niche for pTTSS mutants. Finally, we show that invasin and the chromosomally encoded TTSS were not required for Yptb survival in MLN. In summary, chromosomally encoded factors are sufficient for Yptb replication in the cortex and paracortex of MLN; the pTTSS enables Yersinia to survive within phagocyte-rich areas of lymph nodes, and spread to other tissues.     

Calcitonin inhibits accumulation of cyclic AMP in stimulated peritoneal macrophages from normal rats but not from osteopetrotic (incisors-absent) littermates

Macrophages and osteoclasts derive from related cell lines. In osteopetrotic mutants the function of osteoclasts is greatly reduced compared to that in normal animals or children and macrophage function is variably affected depending upon the mutation. To further explore macrophage function in osteopetrosis we examined the regulation of cyclic AMP production in macrophages from mutants and normal littermates of the osteopetrotic stock incisors-absent (ia) in the rat. Surface stimulation by latex particles of elicited peritoneal macrophages from normal or osteopetrotic (ia) mutant rats caused an identical increase in the accumulation of cyclic AMP. This effect was inhibited in normal animals by coincubation of macrophages with calcitonin (CT) but this inhibition was either absent or less marked in macrophages from mutant littermates. In contrast to human monocytes preincubation of rat macrophages with pertussis toxin did not relieve this inhibition. This implies that rat peritoneal macrophages respond to CT by a different mechanism. These results demonstrate altered macrophage function in osteopetrotic animals and may be functionally related to the reduced CT binding previously described in ia osteoclasts. Furthermore, the coexistence of reduced function of macrophages and osteoclasts in the ia mutation suggests that macrophages and osteoclasts share a common progenitor.     

Identification of genetic loci implicated in the survival of Mycobacterium smegmatis in human mononuclear phagocytes

A luminescence-based procedure that permits the rapid evaluation of the survival of mycobacteria within mononuclear phagocytes was developed and used to screen insertional mutants of Mycobacterium smegmatis for their ability to survive in human monocyte-derived macrophages. Among the 5000 mutants tested, eight mutants were identified that demonstrated impaired intracellular survival in human macrophages but that grew normally in the absence of cells. For each mutant, a portion of the gene interrupted by the transposition event was amplified by ligand-mediated PCR and sequenced. In all cases, the existence of homologous genes of as yet unknown function were identified in the Mycobacteium tuberculosis genome. Complementation of the mutant mycobacterial strains with cosmids containing the homologous loci from M. tuberculosis restored normal intracellular growth in three of the four mutants tested, supporting the idea that these loci contain genes that are important for intracellular survival. This study demonstrates the feasibility of directly screening mutant mycobacterial strains to identify genes coding for activities necessary for the intracellular survival in human mononuclear phagocytes, an important initial step in the identification of potential targets for new therapeutic agents.     

Molecular analysis of myc gene mutants

We describe the generation and characterization of a series of deletion mutants of the avian acute leukaemia virus MC29 which allow the study of the function of the myc in transformation of quail embryo fibroblasts in vitro and tumour induction in vivo. These mutants, which are deleted in the 3' portion of the myc gene, fail to transform macrophages in vitro or induce tumours in vivo but are still able to transform morphologically fibroblasts. From one of these mutants a 'recovered' MC29 virus was generated which, like wild type MC29, transformed fibroblasts and macrophages in vitro. When tested in vivo this virus induced lymphomas of T and B cells rather that the endotheliomas induced by wild type MC29. This system allows us to investigate another question which is the mechanism by which the virus (or oncogene it contains) preferentially transforms one cell type.     

Phylogenetic lineage and pilus protein Spb1/SAN1518 affect opsonin-independent phagocytosis and intracellular survival of Group B Streptococcus

Opsonin-independent phagocytosis of Group B Streptococcus (GBS) is important in defense against neonatal GBS infections. A recent study indicated a role for GBS pilus in macrophage phagocytosis (Maisey et al Faseb J 22 2008 1715-24). We studied 163 isolates from different phylogenetic backgrounds and those possessing or lacking the gene encoding the pilus backbone protein, Spb1 (SAN1518, PI-2b) and spb1-deficient mutants of wild-type (WT) serotype III-3 GBS 874391 in non-opsonic phagocytosis assays using J774A.1 macrophages. Numbers of GBS phagocytosed differed up to 23-fold depending on phylogenetic background; isolates possessing spb1 were phagocytosed more than isolates lacking spb1. Comparing WT GBS and isogenic spb1-deficient mutants showed WT was phagocytosed better compared to mutants; Spb1 also enhanced intracellular survival as mutants were killed more efficiently. Complementation of mutants restored phagocytosis and resistance to killing in J774A.1 macrophages. Spb1 antiserum revealed surface expression in WT GBS and spatial distribution relative to capsular polysaccharide. spb1 did not affect macrophage nitric oxide and TNF-alpha responses; differences in phagocytosis did not correlate with N-acetyl d-glucosamine (from GBS cell-wall) according to enzyme-linked lectin-sorbent assay. Together, these findings support a role for phylogenetic lineage and Spb1 in opsonin-independent phagocytosis and intracellular survival of GBS in J774A.1 macrophages.     

The role of macrophages in demyelinating peripheral nervous system of mice heterozygously deficient in p0

Mice heterozygously deficient in the p0 gene (P0(+/-)) are animal models for some forms of inherited neuropathies. They display a progressive demyelinating phenotype in motor nerves, accompanied by mild infiltration of lymphocytes and increase in macrophages. We have shown previously that the T lymphocytes are instrumental in the demyelination process. This study addresses the functional role of the macrophage in this monogenic myelin disorder.In motor nerves of P0(+/)- mice, the number of macrophages in demyelinated peripheral nerves was increased by a factor of five when compared with motor nerves of wild-type mice. Immunoelectron microscopy, using a specific marker for mouse macrophages, displayed macrophages not only in the endoneurium of the myelin mutants, but also within endoneurial tubes, suggesting an active role in demyelination. To elucidate the roles of the macrophages, we crossbred the myelin mutants with a spontaneous mouse mutant deficient in macrophage colony-stimulating factor (M-CSF), hence displaying impaired macrophage activation. In the P0-deficient double mutants also deficient in M-CSF, the numbers of macrophages were not elevated in the demyelinating motor nerves and demyelination was less severe. These findings demonstrate an active role of macrophages during pathogenesis of inherited demyelination with putative impact on future treatment strategies.     

Isolation and characterization of Francisella novicida mutants defective in lipopolysaccharide biosynthesis

In order to identify genes involved in LPS biosynthesis we isolated random mutants generated by transposon insertion in Francisella novicida. The resulting mutant bank yielded mutants with three distinct LPS phenotypes, and three representative mutants were chosen for further study. One mutant that had short O-antigen chains was sensitive to serum; this mutant and one other were more sensitive to killing by deoxycholate than control strains. The third mutant was resistant to deoxycholate killing but slightly sensitive to serum. The three mutants varied in their ability to grow in macrophages. The DNA sequences interrupted by the transposon in two of the three mutants showed similarity to known LPS biosynthetic genes at the deduced amino acid level.     

Salmonella carrier state and biological characteristics of the infectious agent

Salmonella carrier state (42.6%-S. enteritidis and 34.4%-S. dublin) was demonstrated in subjects after acute salmonellosis as well as in healthy persons infected with salmonella as a result of occupational exposure to poultry (8.8% in humans exposed to chickens and 6.1% in those exposed to ducks) and sheep (2.8%). The carrier state was accompanied by intermittent pain in the epigastrium, diminished appetite, diarrhoea etc. Most of the carrier subjects with a history of salmonellosis exhibited, upon rectoromanoscopy, a varying degree of proctosigmoiditsi. The etiological role of S. typhimurium was proved beyond doubt, as well as its ability to cause salmonellosis outbursts, sporadic cases of the disease and the carrier state. When large industrial facilities specializing in poultry processing were investigated, the salmonella carrier state was revealed in practically healthy poultry--in 16% of chickens and 12% of ducks. The salmonella organisms isolated from carrier persons had, with some exceptions, typical properties, being virulent in that they caused death of experimental animals, seeded their internal organs and induced pathogenicity-associated enzymes. Multiple resistance to antibiotics was demonstrated in salmonella isolated from poultry; also determined was its plasmid nature. Pronounced resistance of the above salmonella subtypes to tetracycline-related antibiotics and streptomycin may be due to the fact that these drugs are used in poultry raising.