Louise M. Hafner

Molecular Evidence to Support the Expansion of the Hostrange of Chlamydophila pneumoniae to Include Reptiles as Well as Humans, Horses, Koalas and Amphibians

The Chlamydiales are a family of unique intracellular pathogens that cause significant disease in humans, birds and a wide range of animal hosts. Of the currently recognized species, Chlamydophila (previously Chlamydia) pneumoniae, unlike the other chlamydial species, has been previously considered to be solely a pathogen of humans, causing significant respiratory disease and has also been strongly connected with cardiovascular disease. Here we report the finding that strains of C. pneumoniae are widespread in the environment, being detected by molecular methods in a range of reptiles (snakes, iguanas, chameleons) and amphibians (frogs, turtles). Of particular interest was the finding that genotyping of the chlamydial major outer membrane protein gene in these newly identified C. pneumoniae strains showed that many were genetically very similar, if not identical to the human respiratory strains. Whether these reptilian and amphibian strains of C. pneumoniae are still capable of infecting humans, or crossed the host barrier some time ago, remains to be determined but may provide further insights into the relationship of this common respiratory infection with its human host.

An improved method for the transformation of Lactobacillus strains using electroporation

Because of their widespread industrial and medical importance, there is considerable interest in the manipulation and improvement of Lactobacillus strains using modern genetic engineering techniques. However, most reports have focused on industrial strains and often have resulted in non-reproducible transformation efficiencies. We have developed an optimised protocol for electroporating foreign plasmid DNA into clinical strains of lactobacilli. Treatment of the recipient lactobacilli with either lysozyme, glycine or penicillin improved electrotransformation efficiencies up to 480-fold. A critical step in achieving efficient and reproducible electrotransformation of clinical lactobacilli with the plasmid pSA3 was the requirement for a post-pulse recovery time of 2-3 h, combined with the use of sub-inhibitory concentrations of antibiotics in the selective plates. While pNZ17 transformants also benefited from a post-pulse recovery period, good transformation efficiencies could be achieved when plated directly onto selective concentrations of chloramphenicol. We also observed significant differences in electrotransformation efficiencies between our guinea pig vaginal Lactobacillus isolates (maximum of 4.8 x 10(4) transformants/mu g pNZ17 DNA) and the human L. casei strain ATCC 393 (3.7 x 10(6) transformants/mu g pNZ17 DNA). An optimised procedure for the electroporation of plasmid DNA into lactobacilli is described.

Chlamydia trachomatis infection: host immune responses and potential vaccines

Chlamydia trachomatis causes genital tract infections that affect men, women, and children on a global scale. This review focuses on innate and adaptive immune responses in the female reproductive tract (FRT) to genital tract infections with C. trachomatis. It covers C. trachomatis infections and highlights our current knowledge of genital tract infections, serovar distribution, infectious load, and clinical manifestations of these infections in women. The unique features of the immune system of the FRT will be discussed and will include a review of our current knowledge of innate and adaptive immunity to chlamydial infections at this mucosal site. The use of animal models to study the pathogenesis of, and immunity to, Chlamydia infection of the female genital tract will also be discussed and a review of recent immunization and challenge experiments in the murine model of chlamydial FRT infection will be presented.

Analysis of promoter sequences from Lactobacillus and Lactococcus and their activity in several Lactobacillus species

Abstract. Promoter-active fragments were isolated from the genome of the probiotic organism Lactobacillus rhamnosus strain GG using the promoter-probe vector pNZ272. These promoter elements, together with a promoter fragment isolated from the vaginal strain Lactobacillus fermentum BR11 and two previously defined promoters (Lactococcus lactis lacA and Lactobacillus acidophilus ATCC 4356 slpA), were introduced into three strains of Lactobacillus. Primer-extension analysis was used to map the transcriptional start site for each promoter. All promoter fragments tested were functional in each of the three lactobacilli and a purine residue was used to initiate transcription in most cases. The promoter elements encompassed a 52- to 1140-fold range in promoter activity depending on the host strain. Lactobacillus promoters were further examined by surveying previously mapped sequences for conserved base positions. The Lactobacillus hexamer regions (–35: TTgaca and –10: TAtAAT) closely resembled those of Escherichia coli and Bacillus subtilis, with the highest degree of agreement at the –10 hexamer. The TG dinucleotide upstream of the –10 hexamer was conserved in 26% of Lactobacillus promoters studied, but conservation rates differed between species. The region upstream of the –35 hexamer of Lactobacillus promoters showed conservation with the bacterial UP element.

Development status and future prospects for a vaccine against Chlamydia trachomatis infection.

Chlamydia trachomatis continues to be the most commonly reported sexually transmitted bacterial infection in many countries with more than 100 million new cases estimated annually. These acute infections translate into significant downstream health care costs, particularly for women, where complications can include pelvic inflammatory disease and other disease sequelae such as tubal factor infertility. Despite years of research, the immunological mechanisms responsible for protective immunity versus immunopathology are still not well understood, although it is widely accepted that T cell driven IFN-g and Th17 responses are critical for clearing infection. While antibodies are able to neutralize infections in vitro, alone they are not protective, indicating that any successful vaccine will need to elicit both arms of the immune response. In recent years, there has been an expansion in the number and types of antigens that have been evaluated as vaccines, and combined with the new array of mucosal adjuvants, this aspect of chlamydial vaccinology is showing promise. Most recently, the opportunities to develop successful vaccines have been given a significant boost with the development of a genetic transformation system for Chlamydia, as well as the identification of the key role of the chlamydial plasmid in virulence. While still remaining a major challenge, the development of a successful C. trachomatis vaccine is starting to look more likely.

Effects of inoculating dose on the kinetics of Chlamydia muridarum genital infection in female mice

Chlamydia trachomatis infections have been implicated in problems such as pelvic inflammatory disease and infertility in females. Although there are some studies examining the kinetics of ascending infection, there is limited information on the kinetics of pathology development and cellular infiltrate into the reproductive tissues in relation to the effects of inoculating dose, and a better understanding of these is needed. The murine model of female genital tract Chlamydia muridarum infection is frequently used as a model of human C. trachomatis reproductive tract infection. To investigate the kinetics of ascending genital infection and associated pathology development, female BALB/c mice were intravaginally infected with C. muridarum at doses ranging from 5 × 102 to 2.6 × 106 inclusion forming units. We found that the inoculating dose affects the course of infection and the ascension of bacteria, with the highest dose ascending rapidly to the oviducts. By comparison, the lowest dose resulted in the greatest bacterial load in the lower reproductive tract. Interestingly, we found that the dose did not significantly affect inflammatory cell infiltrate in the various regions. Overall, this data show the effects of infectious dose on the kinetics of ascending chlamydial infection and associated inflammatory infiltration in BALB/c mice.

Immune regulation during chronic visceral leishmaniasis.

Visceral leishmaniasis is a chronic parasitic disease associated with severe immune dysfunction. Treatment options are limited to relatively toxic drugs, and there is no vaccine for humans available. Hence, there is an urgent need to better understand immune responses following infection with Leishmania species by studying animal models of disease and clinical samples from patients. Here, we review recent discoveries in these areas and highlight shortcomings in our knowledge that need to be addressed if better treatment options are to be developed and effective vaccines designed.

Identification and Characterization of the Novel LysM Domain-Containing Surface Protein Sep from Lactobacillus fermentum BR11 and Its Use as a Peptide Fusion Partner in Lactobacillus and Lactococcus

ABSTRACT Examination of supernatant fractions from broth cultures of Lactobacillus fermentum BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep. The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced. Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of Lactobacillus reuteri, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus agalactiae, and Lactobacillus plantarum. The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of Lactobacillus gasseri and Lactobacillus johnsonii. The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins. We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases. Sep was investigated as a heterologous peptide expression vector in L. fermentum, Lactobacillus rhamnosus GG and Lactococcus lactis MG1363. Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the L. fermentum, L. rhamnosus, and L. lactis hosts. Sep as well as the previously described surface protein BspA were used to express and secrete in L. fermentum or L. rhamnosus a fragment of human E-cadherin, which contains the receptor region for Listeria monocytogenes. This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria.

Structural Changes in the Cells of Some Bacteria during Population Growth: A Fourier Transform Infrared—Attenuated Total Reflectance Study

Structural changes occurring in the cells of several bacteria during their growth curves have been investigated by Fourier transform infrared (FT-IR) spectroscopy using the sampling technique of attenuated total reflectance (ATR). Spectra reflect all of the components of the cells, including the cell walls, cell membranes, internal structures, and the cytoplasm. The bacteria studied were Bacillus stearothermophilus, Halobacterium salinarium, Halococcus morrhuae, and Acetobacter aceti. All species showed significant spectral changes during their growth curves, indicating structural changes in the cells during increases in cell numbers. The major change for B. stearothermophilus was in the lipid content, which was at a maximum during the exponential phase of the growth curve. For the halophiles H. salinarium and H. morrhuae, the major change was that the concentration of sulfate ion in the cells varied during the growth curve and was at a maximum during the mid-part of the exponential phase of the growth curve. A. aceti cells showed increasing polysaccharide content during the growth curve as well as maximum lipid content during the exponential phase of growth.

Peptide surface display and secretion using two LPXTG-containing surface proteins from Lactobacillus fermentum BR11.

ABSTRACT A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L. fermentum BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae, while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri. It was shown that multiple copies of mlp exist in the genome of L. fermentum BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L. fermentum. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His6). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His6 and CFTR peptides or His6 peptide, respectively. Therefore, these newly described proteins from L. fermentum BR11 have potential as protein production and targeting vectors.