Page 1 of 41 Journal of the American Chemical Society 1 2 3 Chemometric Analysis of Bacterial Peptidoglycan Reveals Atypical Modifications which 4 5 6 Empower the Cell Wall against Predatory Enzymes and Fly Innate Immunity 7 8 Akbar Espaillat , Oskar Forsmo , Khouzaima El Biari §, Rafael Björk , Bruno Lemaitre , Johan 9 10 § ^ * 11 Trygg , Francisco Javier Cañada , Miguel A. de Pedro , and Felipe Cava 12 13 14 15 Laboratory for Molecular Infection Medicine Sweden, Department of Molecular Biology, 16 17 18 Umeå Centre for Microbial Research, Umeå University, 90187 Umeå, Sweden. 19 20 §Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro 21 22 de Maeztu 9, 28040 Madrid, Spain. 23 24 25 Department of Chemistry. Umeå University, 90187 Umeå, Sweden. 26 27 Global Health Institute, Swiss Federal Institute of Technology, Station 19, CH1015 Lausanne, 28 29 30 Switzerland. 31 32 ^Centro de Biología Molecular “Severo Ochoa”, Universidad Autónoma de MadridConsejo 33 34 Superior de Investigaciones Científicas, Madrid 28049. Spain. 35 36 37 38 39 *To whom correspondence should be addressed. 40 41 Email: [email protected] 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 1 Journal of the American Chemical Society Page 2 of 41 1 2 3 Abstract 4 5 6 Peptidoglycan is a fundamental structure for most bacteria. It contributes to the cell 7 8 morphology and provides cell wall integrity against environmental insults. While several studies 9 10 11 have reported a significant degree of variability in the chemical composition and organization of 12 13 peptidoglycan in the domain Bacteria, the real diversity of this polymer is far from fully 14 15 explored. This work exploits rapid ultraperformance liquid chromatography and multivariate 16 17 18 data analysis to uncover peptidoglycan chemical diversity in the Class Alphaproteobacteria, a 19 20 group of Gram negative bacteria which are highly heterogeneous in terms of metabolism, 21 22 morphology and lifestyles. Indeed, chemometric analyses revealed novel peptidoglycan 23 24 25 structures conserved in Acetobacteri a: amidation at the a( L)carboxyl of mesodiaminopimelic 26 27 acid and the presence of muropeptides crosslinked by (13) LAla D(meso)diaminopimelate 28 29 30 crosslinks. Both structures are growthcontrolled modifications which influence sensitivity to 31 32 Type VI secretion system peptidoglycan endopeptidases and recognition by the Drosophila 33 34 innate immune system, suggesting relevant roles in the environmental adaptability of these 35 36 37 bacteria. Collectively our findings demonstrate the discriminative power of chemometr ic tools on 38 39 large cell wallchromatographic datasets to discover novel peptidoglycan structural properties in 40 41 bacteria. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 2 Page 3 of 41 Journal of the American Chemical Society 1 2 3 Introduction 4 5 6 One of the defining characteristics of bacteria is the presence of a peptidoglycan layer as a 7 8 critical component of the cell envelope 1. In Gram negative bacteria, a thin peptidoglycan layer 9 10 11 (sacculus) lies in the periplasm between the cytoplasmic and outer membranes (Figure 1A). In 12 13 contrast, Gram positive bacteria contain one thick, multilayered peptidoglycan which envelopes 14 15 the cytopla smic membrane. The canonical monomeric subunit consists of the disaccharide 16 17 18 pentapeptide N- acetylglucosamine b(1®4) Nacetylmuramic acid LAla DGlu()(diamino 19 20 acid) DAla DAla, where mesodiaminopimelic acid and Llysine are the more frequent diamino 21 22 acids of Gram negative and Gram positive acteria, respectively 1. Monomers are converted into 23 24 25 linear polymers by means of b(1®4) glycosidic bonds, and then linear polymers are covalently 26 27 linked by means of peptide bridges between the peptide moieties. The final result is a netlike 28 29 macromolecule which encloses the cell body. Further metabolic activities result in a series of 30 31 32 modifications in the chemical nature of peptidoglycan subunits, and on the relative proportions 33 34 of the different subunits 1. The peptidoglycan sacculus plays crucial roles defining cell shape and 35 36 37 preserving bacterial cell integrity. Its crucial role becomes apparent when peptidoglycan 38 39 synthesis is inhibited by antibiotics (e.g. penicillins), which cause morphological alterations, cell 40 41 lysis and death 2. 42 43 44 Since the cell wall is a barrier between the environment and the cell, it seems quite rational to 45 46 47 expect both adaptive and evolutionary variability in this structure amongst the many existing 48 49 bacterial species. In fact, even the low resolution techniques (e.g. thin layer chromatography 3) 50 51 52 available in the early days of cell wall research pointed out substantial peptidoglycan variability. 53 54 Introduction of high resolution techniques such as HighPerformance Liquid Chromatography 55 56 (HPLC) and its recently improved “UltraPerformance” version (UPLC) 4, revealed new levels 57 58 59 60 ACS Paragon Plus Environment 3 Journal of the American Chemical Society Page 4 of 41 1 2 3 of complexity demonstrating the dynamic nature of peptidoglycan structure and its adaptive 4 5 5,6 6 growthphase dependent peptidoglycan plasticity . Uncovering the breadth of peptidoglycan 7 8 structural variability will lead to a better understanding of cell wall biology in nature, in 9 10 11 particular its role in environmental adaptation and signaling. However, an in depth exploration of 12 13 peptidoglycan variability requires the analysis of large numbers of samples and the application of 14 15 chemometric tools to both, identify novel peptidoglycan traits and define clusters of organisms 16 17 18 sharing these features. In other words, an “omic” approach conceptually similar to proteomics or 19 20 metabolomics. 21 22 23 Cell wall structural variability might be an important element in the bacterial strategy to 24 25 26 adapt to hostile environments, as are most natural habitats. For instance, many Gram negative 27 7 28 bacteria have devised mechanisms to outcompete cohabitating species by releasing or injecting, 29 30 via type VI secretion systems (T6SS) 8, peptidoglycanhydrolases. It has been suggested that 31 32 33 some bacteria might escape by modifying the structure or composition of their peptidoglycan 34 35 target 8a , i.e. chemical modifications 9 and changes to the crosslink positions 10 . In higher 36 37 organisms, peptidoglycan recognition proteins of the innate immune system provide an 38 39 40 antibacterial defense mechanism targeting the cell wall. Because these proteins recognize highly 41 42 conserved motifs in the peptidoglycan, some bacteria avoid recognition through specific 43 44 modifications such as peptidoglycan deacetylation which mitigates host immune detection and 45 46 11 47 thus favors pathogen persistence . 48 49 Here we report the development and application of chemometric tools to the analysis of 50 51 52 peptidoglycan in representative species of the Gram negative bacterial class Alphaproteobacteria, 53 12 54 one of the 6 classes which make up the phylum Proteobacteria . Alphaproteobacteria are 55 56 appropriate sources for cell wall studies aiming at finding new structural variability because of 57 58 59 60 ACS Paragon Plus Environment 4 Page 5 of 41 Journal of the American Chemical Society 1 2 3 their high diversity in terms of metabolism, morphology, environmental niches and lifestyles 4 5 13,14 6 spanning from freeliving bacteria to symbionts and intracellular pathogens . The general 7 8 suitability of this method offers a rapid pipeline to uncover novel peptidoglycan traits in large 9 10 11 datasets. Multivariate data analysis of peptidoglycan UPLC spectra revealed three clusters, one 12 13 of them exclusively comprising members of the family Acetobacteraceae, a family in the 14 15 Alphaproteobacterial order Rhodospirillales characterized by the ability of its members to 16 17 15 18 produce acetic acid and grow at low pH . The cell wall of these bacteria is characterized by the 19 20 presence of previously undescribed muropeptides amidated at the a( L)carboxyl group of meso 21 22 LD 23 diaminopimelic acid (DAP), and crosslinked through (13) peptide bridges. Functional 24 25 studies showed that these new muropeptides are poor substrates for Type VI secretion system 26 27 (T6SS) endopeptidases from the potentially coinhabitant bacteria Pseudomonas aeruginosa and 28 29 30 Acidovorax citrulii. Furthermore, amidation at the carboxyl of DAP reduces the ability of 31 32 peptidoglycan to elicit the innate immune response in the vinegar fly Drosophila melanogaster , a 33 34 natural host for Acetobacteria. 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 5 Journal of the American Chemical Society Page 6 of 41 1 2 3 Results 4 5 6 7 8 Multivariate statistical analysis of peptidoglycan composition reveals three clusters in 9 10 11 Alphaproteobacteria 12 13 As peptidoglycan chemical structure is inferred from muropeptide chromatographic 14 15 profiles, a clustering analysis was expected to reveal the subjacent compositional variability in 16 17 18 large numbers of datasets. Therefore, we selected 20 different species representing five 19 20 (Caulobacterales, Sphingomonadales, Rhodospirillales, Rhodobacterales and Rhizobiales) of the 21 22 six different Orders of the Class Alphaproteobacteria (Table S1). We did not include species 23 24 25 from Order Rickettsiales due to their growth requirements. Peptidoglycan muropeptide 26 27 composition from stationary phase cultures was analyzed by means of ultraperformance liquid 28 29 chromatography (UPLC) (Figure S1) and its variability was assessed by hierarchical cluster 30 31 16 32 analysis (HCA) using principal component analysis (PCA). Both, clustering and interrelations 33 34 were calculated according to the presence (or absence) and relative abundances of “peaks”, in the 35 36 37 UV 204 /retention time ( tR) records.