Erin R. Shanahan

TLR2-targeted secreted proteins from Mycobacterium tuberculosis are protective as powdered pulmonary vaccines

Despite considerable research efforts towards effective treatments, tuberculosis (TB) remains a staggering burden on global health. Suitably formulated sub-unit vaccines offer potential as safe and effective generators of protective immunity. The Mycobacterium tuberculosis antigens, cutinase-like proteins (Culp) 1 and 6 and MPT83, were conjugated directly to the novel adjuvant Lipokel (Lipotek Pty Ltd), a TLR2 ligand that delivers antigen to immune cells in a self-adjuvanting context. Protein-Lipokel complexes were formulated as dry powders for pulmonary delivery directly to the lungs of mice by intra-tracheal insufflation, leading to recruitment of neutrophils and antigen presenting cell populations to the lungs at 72 h, that persisted at 7 days post immunisation. Significant increases in the frequency of activated dendritic cells were observed in the mediastinal lymph node (MLN) at 1 and 4 weeks after homologous boosting with protein-Lipokel vaccine. This was associated with the increased recruitment of effector CD4(+) and CD8(+) T-lymphocytes to the MLN and systemic antigen-specific, IFN-γ producing T-lymphocyte and IgG responses. Notably, pulmonary immunisation with either Culp1-6-Lipokel or MPT83-Lipokel powder vaccines generated protective responses in the lungs against aerosol M. tuberculosis challenge. The successful combination of TLR2-targeting and dry powder vaccine formulation, together with important practical benefits, offers potential for pulmonary vaccination against M. tuberculosis.

Dyspepsia and the microbiome: time to focus on the small intestine.

We note with interest two recent publications in Gut regarding alterations to the gut microbiome in individuals on proton pump inhibitor (PPI) therapy.1 ,2 With PPI use, both studies observed an increase in oral bacteria in the stool. These changes overlap with those described in patients with cirrhosis of the liver.3 However, very little is known about the impact of this dysbiosis over the length of the GI tract, and how it may link to gut function. While the data from both Jackson et al 1 and Imhann et al 2 are demonstrative of a shift towards oral-associated bacteria, it is important to note that many of these bacteria have also been identified in the stomach and small intestine. Recently, we have shown that although the microbiota found in the healthy duodenum are taxonomically similar to the oral microbiota, their presence and association with the intestinal mucosa is not merely a result of luminal contamination.4 Thus, small intestinal dysbiosis and its potential impact on the gut must also be considered. Compared with studies …

Characterisation of the gastrointestinal mucosa-associated microbiota: a novel technique to prevent cross-contamination during endoscopic procedures

The mucosa‐associated microbiota appears to be highly relevant to host–microbe interactions in the gastrointestinal (GI) tract. Thus, precise characterisation of the mucosa‐associated microbiota may provide important insights for diagnostic and therapeutic development. However, for technical reasons, mucosal biopsies taken during standard endoscopic procedures are potentially contaminated by GI luminal contents.

Isolation of genetically tractable most-wanted bacteria by metaparental mating

Metagenomics has rapidly advanced our inventory and appreciation of the genetic potential inherent to the gut microbiome. However it is widely accepted that two key constraints to further genetic dissection of the gut microbiota and host-microbe interactions have been our inability to recover new isolates from the human gut, and the paucity of genetically tractable gut microbes. To address this challenge we developed a modular RP4 mobilisable recombinant vector system and an approach termed metaparental mating to support the rapid and directed isolation of genetically tractable fastidious gut bacteria. Using this approach we isolated transconjugants affiliated with Clostridium cluster IV (Faecalibacterium and Oscillibacter spp.), Clostridium cluster XI (Anaerococcus) and Clostridium XIVa (Blautia spp.) and group 2 ruminococci amongst others, and demonstrated that the recombinant vectors were stably maintained in their recipient hosts. By a similar approach we constructed fluorescently labelled bacterial transconjugants affiliated with Clostridium cluster IV (including Flavonifractor and Pseudoflavonifractor spp.), Clostridium XIVa (Blautia spp.) and Clostridium cluster XVIII (Clostridium ramosum) that expressed a flavin mononucleotide-based reporter gene (evoglow-C-Bs2). Our approach will advance the integration of bacterial genetics with metagenomics and realize new directions to support a more mechanistic dissection of host-microbe associations relevant to human health and disease.

Cutinase-like protein-6 of Mycobacterium tuberculosis is recognised in tuberculosis patients and protects mice against pulmonary infection as a single and fusion protein vaccine

Infection with Mycobacterium tuberculosis continues to be a leading cause of death in many regions of the world, and control of this disease is hampered by the lack of a safe and effective vaccine. Secreted proteins of M. tuberculosis are an important group of antigens for subunit vaccines which target this infection. We have tested three secreted members of the cutinase-like protein (CULP) family of M. tuberculosis for their potential as protein vaccine antigens. Culp6 elicited a strong T lymphocyte response in M. tuberculosis infected mice, and importantly, in tuberculosis (TB) patients tested. Culp1, Culp2 and Culp6 when delivered as protein vaccines to mice, induced potent IFN-gamma responses which in turn translated into a significant level of protection against aerosol M. tuberculosis infection. A Culp1-6 fusion protein provided an increased level of protection against infection compared to Culp1 or Culp6 alone. The data presented here may indicate that the cell wall-associated, putatively essential protein Culp6, shown here for the first time to be recognised in TB patients, is an attractive candidate for inclusion in future subunit vaccines.

Influence of cigarette smoking on the human duodenal mucosa-associated microbiota

BackgroundCigarette smoking is a known risk factor in a number of gastrointestinal (GI) diseases in which the microbiota is implicated, including duodenal ulcer and Crohn’s disease. Smoking has the potential to alter the microbiota; however, to date, the impact of smoking on the mucosa-associated microbiota (MAM), and particularly that of the upper GI tract, remains very poorly characterised. Thus, we investigated the impact of smoking on the upper small intestinal MAM. A total of 102 patients undergoing upper GI endoscopy for the assessment of GI symptoms, iron deficiency, or Crohn’s disease, but without identifiable lesions in the duodenum, were recruited. Smoking status was determined during clinical assessment and patients classified as current (n = 21), previous smokers (n = 40), or having never smoked (n = 41). The duodenal (D2) MAM was profiled via 16S rRNA gene amplicon sequencing.ResultsSmoking, both current and previous, is associated with significantly reduced bacterial diversity in the upper small intestinal mucosa, as compared to patients who had never smoked. This was accompanied by higher relative abundance of Firmicutes, specifically Streptococcus and Veillonella spp. The relative abundance of the genus Rothia was also observed to be greater in current smokers; while in contrast, levels of Prevotella and Neisseria were lower. The MAM profiles and diversity of previous smokers were observed to be intermediate between current and never smokers. Smoking did not impact the total density of bacteria present on the mucosa.ConclusionsThese data indicate the duodenal MAM of current smokers is characterised by reduced bacterial diversity, which is partially but not completely restored in previous smokers. While the precise mechanisms remain to be elucidated, these microbiota changes may in some part explain the adverse effects of smoking on mucosa-associated diseases of the GI tract. Smoking status requires consideration when interpreting MAM data.

Letter: investigating the intestinal mucosa-associated microbiota - relevance and potential pitfalls. Authors' reply.

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Mo1798 Sampling of the Mucosal Microbiota in the Upper Gastrointestinal Tract: A Comparison of the Novel Brisbane Aseptic Mucosal Sampling Device Versus Standard Biopsy

Background: Many studies have focused on the fecal microbiota however it is now widely accepted that the gastrointestinal (GI) mucosa associated microbiota, rather than luminal content, is critically important in host-microbe interactions linked to health and disease. Mucosal biopsy is the most common sampling technique used to assess the mucosa associated microbiota. However, normal biopsy devices are designed to take samples for histologic assessment. Thus when biopsies are taken via working channels that are also used to aspirate luminal content it is highly likely that cross contamination occurs, that ultimately questions the validity of these samples. In view of this difficulty we have developed a novel device that allows targeted biopsies to be taken in any segment of the GI tract without cross contamination in an aseptic manner. Methods: Six patients undergoing upper GI endoscopy for iron deficiency were recruited with consent and ethical approval. In pilot experiments various prototypes of a sampling device were tested in vivo. Two final configurations of a sheath that covered miniature biopsy forceps were tested and compared with the normal single-use biopsy forceps (18 samples). Matched duodenal biopsy samples were collected from each patient, transferred individually into RNA later, and subject to gDNA extraction. Amplicon libraries spanning the V6-V8 region of the 16S rRNA gene were constructed, sequenced using the Illumina MiSeq platform, and analysed via the QIIME pipeline. Results: Microbial DNA, representing a diverse community, was observed in all duodenal samples obtained using the aseptic device, indicating this device is effective in sampling mucosa associated organisms present in the duodenum (Chao 1 estimated richness = 386, Goods coverage 99.8%). Assessment of the duodenal microbiota revealed a community dominated by the genera Streptococcus, Prevotella, Veillonella, Neisseria and Porphyromonas. There were substantial differences in the microbiota of samples obtained using the aseptic device and samples obtained with standard biopsy forceps. Only a low level of correlation (Pearsons r<0.6) between the matched biopsy pairs was seen across all bacterial phyla observed. Phylogenetic based (UniFrac) evaluation of beta-diversity revealed the aseptic samples clustered together and showed reduced inter-individual variability compared to those obtained with standard forceps. Conclusions: Sampling of duodenal biopsies with routine biopsy forceps resulted in greater microbial diversity as compared to samples acquired under aseptic conditions. This suggests cross contamination from other sites of the upper GI tract when utilising standard forceps. Based upon our data, aseptic techniques with a specific sampling device should be used to collect samples for studies that aim to define the mucosa associated microbiota.