Isolation of a thermotolerant Paravahlkampfia sp. from lizard intestine: biology and molecular identification

An amoeba was isolated from the intestines of several moribund pink-tongued skinks (lizards), Hemisphaeriodon gerrardi. Unusual features of this isolate were its ability to grow at temperatures of > or = 37 degrees C, and its inability to use Escherichia coli as a food source or to grow axenically on a variety of enriched culture media suitable for other soil amoeba isolates. Growth was abundant, however, on tissue culture cells, with amoebae clearing cell monolayers in approximately 48 h at 37 degrees C. Trophozoites had a vahlkampfiid-like morphology, moving by means of an anterior eruptive pseudopod. Cysts, round to slightly ovoid and lacking exit pores, were formed in culture. Tests for enflagellation of trophic amoebae were negative. Indirect immunofluorescence staining was negative for Naegleria fowleri and Willaertia sp. The isolate was sensitive to azithromycin, but not to amphotericin B, pentamidine isethionate, fluconazole, 5-fluorocytosine, and sulfadiazine. Phylogenetic analysis based on the PCR-amplified small subunit ribosomal DNA, identified the organism as Paravahlkampfia ustiana, an amoeba not previously isolated from either poikilothermic or homeothermic hosts. No evidence of pathology was seen in stained sections of lizard intestine, suggesting that the ameba was part of the normal fauna of the lizard gut. Its diet in the lizard intestine is unknown and the organism may have unusual growth requirements. Thus, P. ustiana joins other soil amoebae that have been isolated from mammals, amphibia, fish, and reptiles, which have the potential of becoming opportunistic pathogens.     

Identification and characterization of an ATP binding cassette L-carnitine transporter in Listeria monocytogenes

We identified an operon in Listeria monocytogenes EGD with high levels of sequence similarity to the operons encoding the OpuC and OpuB compatible solute transporters from Bacillus subtilis, which are members of the ATP binding cassette (ABC) substrate binding protein-dependent transporter superfamily. The operon, designated opuC, consists of four genes which are predicted to encode an ATP binding protein (OpuCA), an extracellular substrate binding protein (OpuCC), and two membrane-associated proteins presumed to form the permease (OpuCB and OpuCD). The operon is preceded by a potential SigB-dependent promoter. An opuC-defective mutant was generated by the insertional inactivation of the opuCA gene. The mutant was impaired for growth at high osmolarity in brain heart infusion broth and failed to grow in a defined medium. Supplementation of the defined medium with peptone restored the growth of the mutant in this medium. The mutant was found to accumulate the compatible solutes glycine betaine and choline to same extent as the parent strain but was defective in the uptake of L-carnitine. We conclude that the opuC operon in L. monocytogenes encodes an ABC compatible solute transporter which is capable of transporting L-carnitine and which plays an important role in osmoregulation in this pathogen.     

Survival of Campylobacter jejuni in co-culture with Acanthamoeba castellanii: role of amoeba-mediated depletion of dissolved oxygen

Campylobacter jejuni is a major cause of infectious diarrhoea worldwide but relatively little is known about its ecology. In this study, we examined its interactions with Acanthamoeba castellanii, a protozoan suspected to serve as a reservoir for bacterial pathogens. We observed rapid degradation of intracellular C.jejuni in A.castellanii 5 h post gentamicin treatment at 25°C. Conversely, we found that A.castellanii promoted the extracellular growth of C.jejuni in co-cultures at 37°C in aerobic conditions. This growth-promoting effect did not require amoebae - bacteria contact. The growth rates observed with or without contact with amoeba were similar to those observed when C.jejuni was grown in microaerophilic conditions. Preconditioned media prepared with live or dead amoebae cultivated with or without C.jejuni did not promote the growth of C.jejuni in aerobic conditions. Interestingly, the dissolved oxygen levels of co-cultures with or without amoebae - bacteria contact were much lower than those observed with culture media or with C.jejuni alone incubated in aerobic conditions, and were comparable with levels obtained after 24 h of growth of C.jejuni under microaerophilic conditions. Our studies identified the depletion of dissolved oxygen by A.castellanii as the major contributor for the observed amoeba-mediated growth enhancement.     

Sequential cold-sensitive mutations in Aspergillus fumigatus.

Mutants of thermotolerant fungus Aspergillus fumigatus I-21 (ATCC 32722) unable to grow at 37 degrees C were sought. Cold-sensitive mutants were enriched from progeny spores of gamma-irradiated conidia by two or more incubations at various nonpermissive temperatures alternating with filtrations through chessecloth. The approximate minimum, optimum, and maximum growth temperatures of the parent were 12, 40, and 50 degrees C, respectively. Mutants unable to grow at 37 degrees C were not successfully isolated directly from the wild type. A mutant unable to grow at 25 degrees C was isolated and mutations further increasing the cold sensitivity by increments of 3-5 degrees C were found to occur. Mutants completely unable to grow at 37 degrees C were obtained by five sequential mutations. All mutants grew as fast as the wild-type parent at 45 degrees C and higher. Each mutant produced revertants able to grow not only at the nonpermissive temperature used for its isolation but also at lower temperatures.     

Requirement of gene fadD33 for the growth of Mycobacterium tuberculosis in a hepatocyte cell line

Gene fadD33 of Mycobacterium tuberculosis, one of the 36 homologues of gene fadD of Escherichia coli identified in the M. tuberculosis genome, predictively encodes an acyl-CoA synthase, an enzyme involved in fatty acids metabolism. The gene is underexpressed in the attenuated strain M. tuberculosis H37Ra relative to virulent H37Rv and plays a role in M. tuberculosis virulence in BALB/c mice by supporting mycobacterial replication in the liver. In the present paper, we investigated the role of fadD33 expression in bacterial growth within the hepatocyte cell line HepG2, as well as in human monocyte-derived THP-1 cells and peripheral blood mononuclear cells. M. tuberculosis H37Rv proved able to grow within HepG2 cells, while the intracellular replication of M. tuberculosis H37Ra was markedly impaired; complementation of strain H37Ra with gene fadD33 restored its replication to the levels of H37Rv. Moreover, disruption of gene fadD33 by allelic exchange mutagenesis reduced the intracellular growth of M. tuberculosis H37Rv, and complementation of the fadD33-disrupted mutant with gene fadD33 restored bacterial replication. Conversely, fadD33 expression proved unable to influence M. tuberculosis growth in human phagocytes, as fadD33-disrupted M. tuberculosis H37Rv mutant, as well as fadD33-complemented M. tuberculosis H37Ra, grew within THP-1 cells and peripheral monocytes basically at the same rates as parent H37Rv and H37Ra strains. The results of these experiments indicate that gene fadD33 expression confers growth advantage to M. tuberculosis in immortalized hepatocytes, but not in macrophages, thus emphasizing the importance of fadD33 in liver-specific replication of M. tuberculosis.     

Infection of Acanthamoeba castellanii by Chlamydia pneumoniae.

Chlamydia pneumoniae is an intracellular respiratory pathogen, which, similar to Legionella, might have developed mechanisms to escape the intracellular bactericidal activity of both human host cells and amoeba. We therefore investigated the intracellular growth and survival of C. pneumoniae in Acanthamoeba castellanii by using cell culture, immunofluorescence microscopy, and electron microscopy. A castellanii was incubated with purified elementary bodies of C. pneumoniae TW 183 at a concentration of 10(6) inclusion-forming units (IFU)/ml to give a ratio of approximately 1 IFU of C. pneumoniae per amoeba. Quantitative determination of chlamydial growth within A. castellanii revealed viable and infective C. pneumoniae in the range of 10(4) to 10(5) IFU/ml between days 7 and 14 postinfection. Immunofluorescence analysis and transmission electron microscopy with subsequent immunogold staining confirmed evidence of infection of the amoebae by C. Pneumoniae and additionally revealed that C. pneumoniae entered the typical growth cycle. Our results show that amoebae allow the survival of C. pneumoniae, suggesting that amoebae may serve as an additional reservoir for Chlamydia or Chlamydia-related organisms.     

Mutations in cognate genes of Saccharomyces cerevisiae hsp70 result in reduced growth rates at low temperatures.

Expression of two Saccharomyces cerevisiae genes (YG101 and YG103) that are related to the gene encoding inducible 70K protein (hsp70) is repressed upon heat shock. Mutations of the two genes were constructed in vitro and substituted into the yeast genome in place of the wild-type alleles. No phenotypic effect of single mutations of either gene was detected. However, cells containing both YG101 and YG103 mutations showed altered growth properties; double-mutation cells possess an optimal growth temperature of 37 degrees C rather than 30 degrees C and grow increasingly poorly as the temperature is lowered. Mutations of two other members of this hsp70-related multigene family, YG100 and YG102, have been analyzed (E. A. Craig and K. Jacobsen, Cell 38:841-849, 1984). Cells containing both YG100 and YG102 mutations cannot form colonies at 37 degrees C. Fusions between the YG101 and YG102 promoter regions and the YG100 and YG101 structural genes, respectively, were constructed. The YG101 promoter-YG100 structural gene fusion was not able to restore normal growth properties to the yg101- yg103- mutant. Also, yg100- yg102- cells containing the YG102 promoter-YG101 structural gene fusion were unable to grow at 37 degrees C. Failure of the protein products of related genes to rescue the relative cold sensitivity of growth suggests that members of the hsp70 multigene family are functionally distinct.     

Effects of an isogenic Zn-metalloprotease-deficien mutant of Legionella pneumophila in a guinea-pig pneumonia model

To determine the effects, if any, of the Zn-metallo-protease on virulence of Legionella pneumophila infection, an isogenic mutant deficient in protease (encoded by the proA gene) was tested in an Acantha-moeba cell model, in guinea-pig macrophages, and in a guinea-pig pneumonia model. The cloned proA gene was completely inactivated by insertion of a kanamycin-resistance cassette into the protease gene of L. pneumophila AA100. This mutated gene was then introduced into the L. pneumophila chromosome by allelic exchange to form the isogenic ProA mutant AA200. AA200 showed no difference in its ability to enter, survive, or grow in Acanthamoeba and explanted guinea-pig macrophages; neither light nor electron microscopy revealed morphological differences in the eukaryotic cells infected with the protease mutant or the wild-type strains. The proA gene was found to be expressed in L. pneumophila during intracellular growth in amoebae by measuring the light produced from a truncated luxC gene fusion with the proA promoter. Virulence of the protease mutant was attenuated when tested in a guinea-pig model of infection employing the intratracheal Inoculation method. AA200 was slower to cause death, grew to lower numbers in the lungs, resulted in less necrotic debris and a larger macrophage infiltrate, and was more likely to be found in association with macrophage vacuoles than the parent strain.     

Properties of the imidazolylacetolphosphate aminotransferase produced in a mutant demonstrating no apparent genetic involvement of the structural gene.

Genetic studies with strain hisBH22 of Salmonella typhimurium indicate it contains a deletion within the histidine operon involving part of the hisH gene and all of the hisB gene, but not extending into the adjacent hisC gene which is adjacent to hisB. However, the specific activity of the hisC product, imidazolylacetolphosphate aminotransferase (EC 2.6.1.9), in this strain is only 10 to 15% of that found in extracts from other mutants with a normal hisC gene. We have examined the rate of aminotransferase synthesis in this mutant and we find that the rate of synthesis of aminotransferase activity is low in mutant hisBH22, but the rate increases as the temperature of growth is lowered from 37 to 23 C. The low rate of enzyme accumulation is not due to holoenzyme instability at 37 C but instead is due to apoenzyme instability at this temperature. By transducing the hisBH22 marker into a pyridoxine auxotroph and derepressing the histidine operon under conditions where the intracellular concentration of pyridoxal phosphate would be expected to be low, we were able to demonstrate significant apoenzyme production only at the lower temperature. We suggest that the explanation for low aminotransferase specific activity at 37 C is due to the presence of reduced numbers of catalytically active units caused by normal production of an unstable mutant apoenzyme with only approximately 15% of the molecules being activated to holoenzyme. The holoenzyme from strain hisBH22 is stable during growth of this strain at 37 C.     

Formation of D-alanyl-lipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes

The dlt operon of Gram-positive bacteria comprises four genes (dltA, dltB, dltC and dltD) that catalyse the incorporation of D-alanine residues into the cell wall-associated lipoteichoic acids (LTAs). In this work, we characterized the dlt operon of Listeria monocytogenes and constructed a D-Ala-deficient LTA mutant by inactivating the first gene (dltA) of this operon. The DltA- mutant did not show any morphological alterations and its growth rate was similar to that of the wild-type strain. However, it exhibited an increased susceptibility to the cationic peptides colistin, nisin and polymyxin B. The virulence of the DltA- mutant was severely impaired in a mouse infection model (4 log increase in the LD50) and, in vitro, the adherence of the mutant to various cell lines (murine bone marrow-derived macrophages and hepatocytes and a human epithelial cell line) was strongly restricted, although the amounts of surface proteins implicated in virulence (ActA, InlA and InlB) remains unaffected. We suggest that the decreased adherence of the DltA- mutant to non-phagocytic and phagocytic cells might be as a result of the increased electronegativity of its charge surface and/or the presence at the bacterial surface of adhesins possessing altered binding activities. These results show that the D-alanylation of the LTAs contributes to the virulence of the intracellular pathogen L. monocytogenes.     

Differential growth of Legionella pneumophila strains within a range of amoebae at various temperatures associated with in-premise plumbing

Aims: The potential effect of in‐premise plumbing temperatures (24, 32, 37 and 41°C) on the growth of five different Legionella pneumophila strains within free‐living amoebae (Acanthamoeba polyphaga, Hartmannella vermiformis and Naegleria fowleri) was examined. Methods and Results: Compared with controls that actively fed on Escherichia coli prey, when Leg. pneumophila was used as prey, strains Lp02 and Bloomington‐2 increased in growth at 30, 32 and 37°C while strains Philadelphia‐1 and Chicago 2 did not grow at any temperature within A. polyphaga. Strains Lp02, Bloomington‐2 and Dallas 1E did not proliferate in the presence of H. vermiformis nor did strain Philadelphia‐1 in the presence of N. fowleri. Yet, strain Bloomington‐2 grew at all temperatures examined within N. fowleri, while strain Lp02 proliferated at all temperatures except 41°C. More intriguing, strain Chicago 2 only grew at 32°C within H. vermiformis and N. fowleri suggesting a limited temperature growth range for this strain. Conclusions: Identifying the presence of pathogenic legionellae may require the use of multiple host amoebae and incubation temperatures. Significance and Impact of the Study: Temperature conditions and species of amoeba host supported in drinking water appear to be important for the selection of human‐pathogenic legionellae and point to future research required to better understand Legionella ecology.     

DNA sequence and complementation analysis of a mutation in the rplX gene from Escherichia coli leading to loss of ribosomal protein L24.

A mutation in Escherichia coli leads to the loss of ribosomal protein L24, severely impaired growth, and a temperature-sensitive phenotype. The mutation was shown to be in rplX, the gene for protein L24, and was due to the alteration of an AAA codon to a TAA stop codon at position 61 in rplX that resulted in a 20-amino acid peptide instead of the 104 amino acids of wild-type L24 protein. rplX genes from three temperature-resistant and fast growing pseudorevertants of the mutant were cloned and sequenced. They were found to have different base substitutions in the TAA codon, resulting in the reappearance of a full-sized protein L24 moiety. Complementation of the slow growth in trans could be achieved with several plasmids containing at least the spc promoter and intact L14 and L24 genes. Plasmids containing genes distal to rplX could further stimulate growth, and the wild type arose when the entire spc operon and the alpha operon were present. In all cases, protein L24 was expressed by the plasmids. Therefore, slow growth could be explained by polarity extending to the alpha operon. However, temperature sensitivity could not be complemented by any of the plasmids in trans, although we found that this phenotype was caused by the mutation in the rplX gene.