Iain C. Sutcliffe

Lipoprotein biogenesis in Gram-positive bacteria: knowing when to hold ‘em, knowing when to fold ‘em

Gram-positive bacterial lipoproteins are a functionally diverse and important class of peripheral membrane proteins. Recent advances in molecular biology and the availability of whole genome sequence data have overturned many long-held assumptions about the export and processing of these proteins, most notably the recent discovery that not all lipoproteins are exported as unfolded substrates through the general secretion pathway. Here, we review recent discoveries concerning the export and processing of these proteins, their role in virulence in Gram-positive bacteria and their potential as vaccine candidates or targets for new antimicrobials.

A phylum level perspective on bacterial cell envelope architecture

Improved understanding of the bacterial phylogenetic tree has allowed the distinction of at least 25 phyla with cultured representatives. This review surveys the diversity of cell envelope types present in these phyla and emphasises that it is important to define bacterial cell envelopes according to whether they have one (monoderm) or two (diderm) cellular membranes and, in the latter case, lipopolysaccharide as well. A comparative genomics approach, facilitated by the recent vast expansion in genome sequence information, is used here to survey the distribution of key lipopolysaccharide biosynthesis enzymes across the bacterial world and to consider alternative diderm cell envelope architectures. These data add to our understanding of microbial diversity and it is notable that the majority of phyla are likely to comprise diderm, lipopolysaccharide containing bacteria. This analysis and a critical review of the literature also suggest that members of the phylum Chloroflexi are typically monoderm.

The molecular basis of Streptococcus equi infection and disease

Streptococcus equi is the aetiological agent of strangles, one of the most prevalent diseases of the horse. The animal suffering and economic burden associated with this disease necessitate effective treatment. Current antibiotic therapy is often ineffective and thus recent attention has focused on vaccine development. A systematic understanding of S. equi virulence, leading to the identification of targets to which protective immunity can be directed, is a prerequisite of the development of such a vaccine. Here, the virulence factors of S. equi are reviewed.

A novel lipoarabinomannan from the equine pathogen Rhodococcus equi. Structure and effect on macrophage cytokine production.

Rhodococcus equi is a major cause of foal morbidity and mortality. We have investigated the presence of lipoglycan in this organism as closely related bacteria, notablyMycobacterium tuberculosis, produce lipoarabinomannans (LAM) that may play multiple roles as virulence determinants. The lipoglycan was structurally characterized by gas chromatography-mass spectrometry following permethylation, capillary electrophoresis after chemical degradation, and 1H and 31P and two-dimensional heteronuclear nuclear magnetic resonance studies. Key structural features of the lipoglycan are a linear α-1,6-mannan with side chains containing one 2-linked α-d-Manpresidue. This polysaccharidic backbone is linked to a phosphatidylinositol mannosyl anchor. In contrast to mycobacterial LAM, there are no extensive arabinan domains but single terminal α-d-Araf residue capping the 2-linked α-d-Manp. The lipoglycan binds concanavalin A and mannose-binding protein consistent with the presence of t-α-d-Manp residues. We studied the ability of the lipoglycans to induce cytokines from equine macrophages, in comparison to whole cells of R. equi. These data revealed patterns of cytokine mRNA induction that suggest that the lipoglycan is involved in much of the early macrophage cytokine response to R. equi infection. These studies identify a novel LAM variant that may contribute to the pathogenesis of disease caused by R. equi.

Cell envelope composition and organisation in the genus Rhodococcus.

A knowledge of the organisation of the rhodococcal cell envelope is of fundamental importance if the environmental and biotechnological significance of these bacteria are to be understood and succesfully exploited. The genus Rhodococcus belongs to a distinctive suprageneric taxon, the mycolata, which includes among others the genera Corynebacterium, Mycobacterium and Nocardia. Members of this taxon exhibit an unusual complexity in their cell envelope composition and organisation compared to other Gram-positive bacteria. Models that describe the architecture of the mycobacterial cell envelope are extrapolated here to provide a model of the rhodococcal cell envelope. The rhodococcal cell envelope is dominated by the presence of an arabinogalactan cell wall polysaccharide and large 2-alkyl 3-hydroxy branched-chain fatty acids, the mycolic acids, which are covalently assembled into a peptidoglycan–arabinogalactan–mycolic acid matrix. This review further emphasises that the mycolic acids in this complex form the basis of an outer lipid permeability barrier. The localisation and roles of other cell envelope components, notably complex free lipids, lipoglycans, proteins and lipoproteins are also considered.

Introducing a digital protologue: a timely move towards a database-driven systematics of archaea and bacteria

It is self-evident that contemporary biological science has become a database-driven endeavour, ranging from databases serving broad communities (such as Genbank) to those that are more specialised. We have argued previously (Rosselló-Móra 2012; Sutcliffe et al. 2012) that it would greatly benefit Archaeal and Bacterial systematics to create an iterative taxonomic database of Archaeal and Bacterial species, which would provide a summative and evolving repository for information on species characteristics, such as those typically found in the protologues of descriptions of novel genera and species. Indeed, such a database would be a welcome complement to MycoBank, a comparable initiative in fungal taxonomy (Robert et al. 2013), and important databases of microbial nomenclature and identification such as LPSN, LTP and EzTaxon (Yarza et al. 2010; Parte 2014; Yoon et al. 2017). As Editors of Antonie van Leeuwenhoek and Systematic and Applied Microbiology, we are responsible for two journals that effectively publish the descriptions of significant numbers of Archaeal and Bacterial species. Given the importance of getting a database established, we have therefore developed a ‘digital protologue’ database (DPD) to accompany descriptions of novel Archaeal and Bacterial taxa published in these two journals. Having been tested by volunteer authors, we believe the DPD has good functionality and fitness for purpose as a repository for taxonomic data, and we expect to refine and improve its design and interactivity in response to feedback from users. Significant features include accession numbers to relevant sequence databases (which will become still more important as whole genome sequences increasingly accompany descriptions of novel microbial taxa) and the generation of unique TaxoNumbers for each entry (which can also be cited and used for microattribution purposes). Moreover, content is exportable in a tabulated format that can be used as Online Supplementary material to accompany published material. At present the database is focussed on descriptions of Archaeal and Bacterial species although options are available to create entries for the description of genera and Candidatus taxa. As the current version will no doubt evolve, we encourage all users to feed-back to the editors for improvements. R. Rosselló-Móra Marine Microbiology Group, Department of Ecology and Marine Resources, Institut Mediterrani d’Estudis Avançats (CSIC-UIB), E-07190 Esporles, Balearic Islands, Spain

Mannan Chain Length Controls Lipoglycans Signaling via and Binding to TLR2

TLR2 is a pattern-recognition receptor that is activated by a large variety of conserved microbial components, including lipoproteins, lipoteichoic acids, and peptidoglycan. Lipoglycans are TLR2 agonists found in some genera of the phylogenetic order Actinomycetales, including Mycobacterium. They are built from a mannosyl-phosphatidyl-myo-inositol anchor attached to a (α1→6)-linked d-mannopyranosyl chain whose units can be substituted by d-mannopyranosyl and/or d-arabinofuranosyl units. At this time, little is known about the molecular bases underlying their ability to induce signaling via this receptor. We have recently shown that the anchor must be at least triacylated, including a diacylglyceryl moiety, whereas the contribution of the glycosidic moiety is not yet clearly defined. We show herein that lipoglycan activity is directly determined by mannan chain length. Indeed, activity increases with the number of units constituting the (α1→6)-mannopyranosyl backbone but is also critically dependent on the substitution type of the 2-hydroxyl of these units. We thus provide evidence for the definition of a new pattern that includes the nonlipidic moiety of the molecules, most probably as a result of the (α1→6)-mannopyranosyl backbone being a highly conserved structural feature among lipoglycans. Moreover, we demonstrate that lipoglycans can bind cell surface-expressed TLR2 and that their ability to induce signaling might be, at least in part, dictated by their avidity for the receptor. Finally, our data suggest that lipoglycans and lipoproteins have a common binding site. The present results are thus discussed in the light of the recently published crystal structure of a TLR1-TLR2-lipopeptide complex.

Transport of Sugars, Including Sucrose, by the msm Transport System of Streptococcus mutans:

The range of substrates transported by the sugar-binding protein-dependent msm (multiple sugar metabolism) system of S. mutans was investigated. By determining the ability of unlabeled sugar to compete with radiolabeled melibiose transport, we have demonstrated that the transported sugars included a number of carbohydrates structurally related to raffinose. A model accommodating these results has been devised which accounts for the sugars transported by the msm transport system. Competition with radiolabeled melibiose transport indicated sucrose to be an msm substrate. This was confirmed by examination of uptake of radiolabeled sucrose in scrAB mutants lacking the sucrose-specific phosphotransferase system.

Characterisation of a lipomannan lipoglycan from the mycolic acid containing actinomycete Dietzia maris

Lipoglycans such as the mycobacterial lipoarabinomannans (LAM) are important cell envelope components of actinomycetes. To further our understanding of the diversity of these enigmatic macromolecules the lipoglycan composition of Dietzia maris has been investigated. Phenol-water extraction and hydrophobic interaction chromatography were used to purify a lipoglycan which was unusually small and predominantly lipomannan in nature. The presence of minor levels of arabinose along with components consistent with the presence of a phosphatidylinositol anchor suggest that this lipoglycan is a novel representative of the lipomannan/LAM structural archetype. This was further supported by the observed cross-reaction of the D. maris lipoglycan with an antiserum raised against LAM from Mycobacterium tuberculosis. These findings reveal a previously unsuspected diversity in the lipoglycan composition of the mycolic acid containing actinomycetes and are further discussed in relation to the apparent absence of phosphatidylinositolmannoside glycolipids in D. maris.

Mutation of the Maturase Lipoprotein Attenuates the Virulence of Streptococcus equi to a Greater Extent than Does Loss of General Lipoprotein Lipidation

ABSTRACT Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (ΔprtM138-213, with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (Δlgt190-685). Moreover, mucus production was significantly greater in both wild-type-infected and Δlgt190-685-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the Δlgt190-685 mutant did still exhibit signs of disease. In contrast, only the ΔprtM138-213 mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the Δlgt190-685 mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted.