Antimicrobial Peptides against Listeria monocytogenes : omic approaches and potential biotechnological applications

A Listeria monocytogenes representa uma ameaça, especialmente para pessoas com sistema imunológico fraco, crianças e mulheres grávidas. A Nisina é utilizada na indústria alimentar para comparar surtos de bactérias Gram-positivas que podem pôr em risco a saúde dos consumidores. No entanto, a pesquisa de novos compostos antimicrobianos (AMPs) tornou-se fundamental para contrastar a detecção cada vez mais frequente da resistência à nisina por L. monocytogenes. Neste trabalho, Bacillus velezensis P34 produtor de fengicina um lipopeptídeos cíclicos foi estudado como possível alternativa de AMPs para uso contra L. monocytogenes. O genoma completo de B. velezensis P34 foi investigado quanto à presença de aglomerados de genes de compostos antimicrobianos. Os compostos secretados foram identificados como fengicina A e B, e em menores quantidades bacilomicina L, por meio de análise de espectrometria de massas. A análise proteômica foi realizada para estudar o "proteosurfactoma", incluindo a proteína moonlight de L. monocytogenes tratada com uma concentração subletal de nisina, detectando a inibição do processo de virulência, além da promoção do biofilme como estratégia de resistência bacteriana. Um estudo proteômico adicional permitiu pela primeira vez a reação fisiológica em L. monocytogenes tratada com os AMPs secretados por Bacillus velezensis P34. Neste estudo, uma nanoencapsulação em lipossomas foi avaliada como aplicação biotecnológica. Dentre os principais resultados deste último trabalho, foi observada forte desregulação dos transportadores de manganês com intensa regulação de outros transportadores de metais para a homeostase de íons metálicos, além de forte regulação negativa de proteínas relacionadas com os principais fatores de virulência Prfa, σB e virR. Além disso, foi realizado um estudo de comparação proteômica e lipidômica adicional, no qual os AMPs de nisina e fengicina foram considerados nesta pesquisa. A forte regulação negativa de proteínas e a variação quantitativa dos ácidos graxos do lipidoma da membrana sugeriram fortemente a inibição do biofilme quando tratado com uma concentração subletal de lipopeptídeos de fengicina; enquanto um grupo de proteínas de membrana foi investigado por sua ação na manutenção da possível resistência de membrana aos AMPs. Os resultados sugeriram a estrutura multicelular do biofilme como a principal preocupação quando pequenas quantidades de nisina podem ser utilizadas, além de que o estudo da ação de compostos secretados de Bacillus velezensis P34 sobre L. monocytogenes pode representar um importante ponto de partida para estudos mais aprofundados de fengicina para uso contra Gram-bactérias positivas, incluindo cepas multirresistentes.Listeria monocytogenes poses a threat especially to people with weak immune systems, children, and pregnant women. Nisin is employed in food industries to contrast Gram positive bacteria outbreaks, which could endanger the health of consumers. However, the research of new antimicrobial compounds (AMPs) has become pivotal to contrast the always more frequent detection of L. monocytogenes nisin resistance. In this doctoral dissertation, the Bacillus velezensis P34, producer of fengycin cyclic lipopeptides as possible alternative AMPs, to use against L. monocytogenes, was studied. Bacillus velezensis P34 whole genome was investigated because of the presence of antimicrobial compound gene clusters, and the secreted compounds were identified as fengycin A and B and, in smaller quantities, bacillomycin L through mass spectrometric analysis. Proteomics analysis was carried out to study the “proteosurfactome” including moonlight protein of L. monocytogenes treated with a sub-lethal concentration of nisin detecting the inhibition of the virulence process besides the biofilm promotion as a bacterial resistance strategy. An additional proteomic study permitted, for the first time, to detect the physiological reaction on L. monocytogenes treated with the AMPs secreted by Bacillus velezensis P34; in the latter, a biotechnological application, specifically a liposome nanoencapsulation, was evaluated. Among the main results of this latter work, strong deregulation of the manganese transporters was observed with intense regulation of others metals transporters for the metal ions homeostasis, besides a strong downregulation of proteins related with the main virulence factors Prfa, σB and virR Moreover, an additional proteomic and lipidomic comparison study was performed, nisin and fengycin AMPs were considered in the experimental drawing of this latter. The strong downregulation of proteins and, quantitatively, the variation of membrane lipidome fatty acids strongly suggested the inhibition of biofilm when treated with a sub-lethal concentration of fengycin lipopeptides; whereas a group of membrane proteins was investigated for their action on the maintaining of the possible membrane resistance to AMPs. The results suggested the promotion of biofilm multicellular organization as the main concern when sub-lethal concentration of nisin was used, besides to that the L. monocytogenes responses to the CLPs synthesized by Bacillus velezensis P34 may represent an important start for deeper studies involving the antimicrobial action of fengycin to use against Gram-positive bacteria, including multiresistant strains

Natural pigments of microbial origin

The world demands new solutions and products to be used as dyes for industrial applications. Microbial pigments represent an eco-friendly alternative as they can be produced in large amounts through biotechnological processes and do not present environmental risks, as they are easily decomposable. Moreover, some of these metabolites are recognized for their biological activities, which qualify them for potential uses as food colorants and nutraceuticals, protecting against degenerative diseases related with oxidative stress. Because of their genetic simplicity as compared with plants, microorganisms may be a better source to understand biosynthetic mechanisms and to be engineered for producing high pigment yields. Despite the origin of the pigmented microorganism, it seems very important to develop protocols using organic industrial residues and agricultural byproducts as substrates for pigment production and find novel green strategies for rapid pigment extraction. This review looks for the most recent studies that describe microbial pigments from microalgae, fungi, and bacteria. In particular, the underexploited tools of omics science such as proteomics and metabolomics are addressed. The use of techniques involving mass spectrometry, allows to identify different protein and metabolite profiles that may be associated with a variety of biotechnologically-relevant pathways of pigment synthesis

Deciphering the chemical dialogue between Bacillus and pathogenic fungi.

In nature, bacteria frequently form bacterial communities known as biofilms, where cells are embedded within an extracellular matrix (ECM) that provides protection against external aggressions or facilitates the efficient uptake and utilization of available resources. Interactions with other microbes can notably alter the community structure and, consequently, the nature of the relationship with the environment1. Previous studies of our laboratory have demonstrated the significance of biofilm formation in the antagonistic interaction between Bacillus and the phytopathogenic fungi Botrytis in the melon phyllosphere2. Our hypothesis is that the ECM plays a complementary role to the structural aspects of this antagonistic interaction. In this study, we dissect how the different components of Bacillus ECM mediate the adhesion of bacterial cells to Botrytis hyphae, which could enhance the efficient release of antifungal metabolites. We also describe how several purified components of the ECM and specific secondary metabolites of Bacillus participate in the chemical communication between Bacillus and Botrytis, thereby altering the physiology and metabolism of Botrytis. Our findings unveil that during this antagonistic interaction, Botrytis secrets different oxylipins, defence molecules capable of killing Bacillus. In response, Bacillus increases the production of several secondary metabolites, which appears to have antifungal effects. Our results underscore the urgency of further investigation of these interactions with the aim of identifying and describing adaptation processes that either lead to the exclusion or coexistence of two initially antagonistic microorganisms.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Marine bacteria as source of antimicrobial compounds

The marine environment encompasses a huge biological diversity and can be considered as an underexplored location for prospecting bioactive molecules. In this review, the current state of art about antimicrobial molecules from marine bacteria has been summarized considering the main phylum and sources evolved in a marine environment. Considering the last two decades, we have found as most studied group of bacteria producers of substances with antimicrobial activity is the Firmicutes phylum, in particular strains of the Bacillus genus. The reason for that can be attributed to the difficult cultivation of typical Actinobacteria from a marine sediment, whose members are the major producers of antimicrobial substances in land environments. However, a reversed trend has been observed in recent years with an increasing number of reports settling on Actinobacteria. Great diversity of chemical structures have been identified, such as fijimicyns and lynamicyns from Actinomycetes and macrolactins produced by Bacillus

Listeria monocytogenes exposed to antimicrobial peptides displays differential regulation of lipids and proteins associated to stress response

With the onset of Listeria monocytogenes resistance to the bacteriocin nisin, the search for alternative antimicrobial treatments is of fundamental importance. In this work, we set out to investigate proteins and lipids involved in the resistance mechanisms of L. monocytogenes against the antimicrobial peptides (AMPs) nisin and fengycin. The effect of sub-lethal concentrations of nisin and lipopeptide fengycin secreted by Bacillus velezensis P34 on L. monocytogenes was investigated by mass spectrometry-based lipidomics and proteomics. Both AMPs caused a differential regulation of biofilm formation, confirming the promotion of cell attachment and biofilm assembling after treatment with nisin, whereas growth inhibition was observed after fengycin treatment. Anteiso branched-chain fatty acids were detected in higher amounts in fengycin-treated samples (46.6%) as compared to nisin-treated and control samples (39.4% and 43.4%, respectively). In addition, a higher relative abundance of 30:0, 31:0 and 32:0 phosphatidylglycerol species was detected in fengycin-treated samples. The lipidomics data suggest the inhibition of biofilm formation by the fengycin treatment, while the proteomics data revealed downregulation of important cell wall proteins involved in the building of biofilms, such as the lipoteichoic acid backbone synthesis (Lmo0927) and the flagella-related (Lmo0718) proteins among others. Together, these results provide new insights into the modification of lipid and protein profiles and biofilm formation in L. monocytogenes upon exposure to antimicrobial peptides

Two-Dimensional Liquid Chromatography Tandem-Mass Spectrometry Untangles the Deep Metabolome of Marine Dissolved Organic Matter

Dissolved organic matter (DOM) is one of the most complex chemical mixtures and plays a central role in biogeochemical cycles across our ecosphere. Despite its importance, DOM remains poorly understood at the molecular level. Over the last decades, significant efforts have been made to decipher the chemical composition of DOM by high-resolution mass spectrometry (HRMS) and liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). Yet, the complexity and high degree of non-resolved isomers still hamper the full structural analysis of DOM. To overcome this challenge, we adapted a two-dimensional (2D) LC approach consisting of two reversed-phase dimensions with orthogonal pH, followed by MS/MS data acquisition and molecular networking. The 2D chromatography approach mitigates the complexity of DOM, enhancing both the quality of MS/MS spectra and spectral annotation rates. Applying our approach to analyze coastal surface DOM from Southern California (USA), we annotated in total more than 600 structures via MS/MS spectrum matching, which was up to 90% more than in iterative 1D LC-MS/MS analysis with the same total run time. Our data provide an unprecedented view into the molecular composition of coastal DOM, highlighting the potential of 2D LC-MS/MS approaches to decipher ultra-complex mixtures

Evaluation of Data-Dependent MS/MS Acquisition Parameters for Non-Targeted Metabolomics and Molecular Networking of Environmental Samples: Focus on the Q Exactive Platform.

Non-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely used tool for metabolomics analysis, enabling the detection and annotation of small molecules in complex environmental samples. Data-dependent acquisition (DDA) of product ion spectra is thereby currently one of the most frequently applied data acquisition strategies. The optimization of DDA parameters is central to ensuring high spectral quality, coverage, and number of compound annotations. Here, we evaluated the influence of 10 central DDA settings of the Q Exactive mass spectrometer on natural organic matter samples from ocean, river, and soil environments. After data analysis with classical and feature-based molecular networking using MZmine and GNPS, we compared the total number of network nodes, multivariate clustering, and spectrum quality-related metrics such as annotation and singleton rates, MS/MS placement, and coverage. Our results show that automatic gain control, microscans, mass resolving power, and dynamic exclusion are the most critical parameters, whereas collision energy, TopN, and isolation width had moderate and apex trigger, monoisotopic selection, and isotopic exclusion minor effects. The insights into the data acquisition ergonomics of the Q Exactive platform presented here can guide new users and provide them with initial method parameters, some of which may also be transferable to other sample types and MS platforms