Bacteria and bacterial products: Foe and friends to Caenorhabditis elegans

Accepted Manuscript Title: Bacteria and bacterial products: Foe and friends to Caenorhabditis elegans Authors: Fazlurrahman Khan, Saurabh Jain, Sandra Folarin Oloketuyi PII: DOI: Reference:

S0944-5013(18)30365-3 https://doi.org/10.1016/j.micres.2018.06.012 MICRES 26183

To appear in: Received date: Revised date: Accepted date:

30-3-2018 11-6-2018 24-6-2018

Please cite this article as: Khan F, Jain S, Oloketuyi SF, Bacteria and bacterial products: Foe and friends to Caenorhabditis elegans, Microbiological Research (2018), https://doi.org/10.1016/j.micres.2018.06.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Bacteria and bacterial products: Foe and friends to Caenorhabditis elegans Running Head: Interaction between bacteria and C. elegans

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Fazlurrahman Khan*, Saurabh Jain† and Sandra Folarin Oloketuyi†

Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater

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These authors equally contributed

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*Corresponding Author

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Noida-201306, U.P., India

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Email address: [email protected]

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Abstract Caenorhabditis elegans is a model organism for the study of different molecular, biochemical, microbial and immunity-related mechanisms. In its natural habitat, C. elegans survives by feeding

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microorganisms (mainly bacteria), though majorly on Escherichia coli OP50 when grown in the laboratory. Numerous bacteria are shown to influence the lifespan, behavioural responses and innate immunity of C. elegans. The secondary metabolites produced by bacteria have shown to play key role in C. elegans longevity. This behaviour provides insights for potential development of new strategies for the treatment of diseases in other species, including humans. This review

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explains the concept of C. elegans microbiome, different mechanisms employed in its longevity

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and resistance against bacterial pathogens and the effects of various bacteria (both beneficial and

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harmful) as well as their products on the life cycle of C. elegans.

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Keywords: Bacteria, C. elegans, longevity, metabolites

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Introduction Caenorhabditis elegans (C. elegans) is an excellent model organism for the study of different aspects such as host-microbe interaction, longevity, neurological disorders, immunity, DNA

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damages, and apoptosis in different species (Arvanitis et al., 2013; Zhang and Hou, 2013; Alexander et al., 2014), due to its lenient characteristics like easy handling, growth in the laboratory and short lifespan. A study revealed that almost two-third of human proteins possess homologues in the whole genome of C. elegans (Sonnhammer and Durbin, 1997). In a review paper, Tissenbaum (2015) discussed the molecular genetics of C. elegans and also explained its

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several advantages and disadvantages that can help in the study of human aging. C. elegans is a

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free-living bacteriovorus nematode that can be found in different bacterial rich environments, such

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as decaying vegetation, rotten fruits and compost and some invertebrates (Kaplan et al., 2009),

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which gives a continuous exposure towards both pathogenic and non-pathogenic bacterial species. An excellent review explained that C. elegans interacts with bacteria through three mechanisms-

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as source of nutrient, aversive learning through neuronal responses, pathogenicity and host

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resistance (Kim, 2013). These mechanisms may affect its ageing and longevity via secretion of

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metabolites by bacteria, induction of stress and immune response as well as modulation of signaling pathway as shown in Figure 1 (Clark and Hodgkin, 2013; Komura et al., 2013; Lee et

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al., 2015a; Martinez et al., 2015; Kwon et al., 2016). Due to the bacteriovorus property of C. elegans, the nematode is broadly used as a model for

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studying predator-prey interactions. This helps to elucidate the synthesis and secretion of the antimicrobial molecule(s), immunological responses, death of microbe inside the body of worm, and biofilm formation by pathogenic bacteria inside the host (King et al., 2016; Sorathia and Rajadhyaksha, 2016). Several studies showed that C. elegans possesses well-developed 3

chemosensory systems that help in the search for food and sensing a wide range of chemicals in the natural environment (Zhang et al., 2005; Beale et al., 2006; Rengarajan and Hallem, 2016). In the environment, many bacteria possess quorum sensing signaling mechanism for maintaining the

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homeostasis of their own communities (Kaper and Sperandio, 2005) and develop defence mechanism by producing toxins (secondary metabolites) against other bacterial pathogens (Neidig et al., 2011). Bacteria such as Pseudomonas aeruginosa, Bacillus megaterium, and Staphylococcus aureus can be pathogenic towards C. elegans either by producing secondary metabolites, such as toxin, and killing the nematode quickly, or by colonizing in the intestine of C. elegans and

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persistent infection resulting in slow death (Tan et al., 1999; Irazoqui et al., 2010; Kirienko et al.,

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2014). Thus, C. elegans protects itself from bacterial infection by innate immunological responses

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and olfactory avoidance properties (Nicholas and Hodgkin, 2004b; Zhang et al., 2005; Ermolaeva

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and Schumacher, 2014). Escherichia coli OP50 is the well-known food source for C. elegans in the laboratory (Samuel et al., 2016). However, several other bacterial species are identified that

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serve as better food sources thereby enhancing the longevity of C. elegans. The defense signaling

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cascades are involved in the production of several immune effector molecules and reactive oxygen

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species in C. elegans upon interaction with these bacterial species (Kim et al., 2004; Shivers et al., 2010; Ermolaeva and Schumacher, 2014). Several metabolic products/secondary metabolites

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produced by non-pathogenic bacteria are involved in protecting C. elegans from being infected by pathogenic bacteria. Hence understanding the interaction of pathogenic and non-pathogenic

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bacteria or their products with C. elegans at molecular levels will open an opportunity to understand the same in other species including humans. The present review describes the role of different bacterial species and their products in the lifespan of C. elegans.

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C. elegans microbiome and its natural environment Research on the importance of C. elegans in its natural environment is limited and aspects such as nutritional sources and patterns, ecological interaction, reproduction and development that were

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shaped by evolution have become undetectable in the laboratory environment (Clark and Hodgkin, 2016; Samuel et al., 2016; Zhang et al., 2017a). C. elegans is predominantly found in humid temperate regions, proliferate in microbe-dense rotting fruits such as apples, oranges, Opuntia cactus fruit, mushrooms, or vegetation (Tamus communis stem) and persists as stress-resistant dauers in the soil or compost (Andersen et al., 2012; Felix and Duveau, 2012; Samuel et al., 2016;

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Schulenburg and Felix, 2017). Dirksen and colleagues (2016) evaluated the composition of C.

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elegans microbiome from different substrates (invertebrate vector, rotting apples and compost)

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and locations (Portugal, Northern Germany and France). Using MiSeq 16S rRNA genotyping, they

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identified a high abundance of Proteobacteria (Enterobacteriaceae), Sphingomonas, Ochrobactrum, Stenotrophomonas and Pseudomonas in C. elegans naïve microbiome which

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differed from the bacterial composition of the corresponding substrates. Similarly, Berg et al.

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(2016) described the contribution of host specificity and ecological diversity of C. elegans. The

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gut microbiota showed different and less microbial diversity comprising of mostly Enterobacteriaceae, Pseudomonadaceae, Xanthomonadaceae

and Comamonadaceae as

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compared to its environment based on functional evaluation and 16S rRNA gene deep sequencing data. In contrast to artificial laboratory condition, most of these bacteria especially

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Enterobacteriaceae, serve as the main nutritional source which support the growth and metabolism of C. elegans. C. elegans harbours these bacteria in its cuticle or intestine and may be dispersed by animal vectors such as snails, isopods, myriapods, chilopods, and slugs (Frezal and Felix, 2015). Based on these studies, C. elegans encounters Proteobacteria, Bacteroidetes, Firmicutes, 5

and Actinobacteria as the most prevalent bacterial phyla in its natural habitat (Kiontke et al., 2011; Dirksen et al., 2016; Samuel et al., 2016). Thus, a better understanding of the worm’s microbiome such as sensory preference, nutrition and other factors influencing its population growth and

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development would help in development of strategies for preventing infectious diseases. Several research and review articles have been reported on modeling the host-microbiome interactions in C. elegans to understand the composition, diversity and function of the microbiome as well as study the mechanism involved in different biological processes (Gerbaba et al., 2017; Zhang et al., 2017b; Ezcurra, 2018; Jiang and Wang, 2018). The first and simplified model is interaction

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between E. coli OP50 as single food source for C. elegans to identify the effect of specific species

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in host phenotype in relation to aging, immunity, pathogenicity and other specific function(s) in

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the microbiome (Stiernagle, 2006; Samuel et al., 2016). Similarly, a recent report showed the

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comparative study on host-microbe interaction in C. elegans by growing it on native microbiome versus laboratory food bacterium thereby analyzing the protein level differences (Cassidy et al.,

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2018). Other model include the use of defined synthetic experimental microbiome which is a

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representation of the most abundant phylum (Proteobacteria) in native host to study C. elegans

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development and genetics (Dirksen et al., 2016); the use of bacterial metabolic pathways (folate and tryptophan synthesis) to understand the growth, development and aging in C. elegans

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(Cabreiro et al., 2013; Virk et al., 2016); association between bacteria (Streptomyces venezuelae and E. coli) and worm expressing human neuronal genes/proteins to study pathology of microbial

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mediated-neurodegeneration. The effect of host-microbe interaction in C. elegans development also involves different components of bacterial respiratory chain (Zhang et al., 2017b). C. elegans fed with mutant strains of E. coli showed developmental delay which could be attributed to oxidative stress induced by bacteria (Govindan et al., 2015). Whittaker et al. (2016) studied the 6

inhibitory effect of benzimidazoles and albendazole on C. elegans and Enterobacteriaceae interaction to evaluate the role of worm's gut microbiota in drug resistance development. Similarly, a host-microbe interaction model system was developed by Garcia-Gonzalez et al. (2017) to study

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the effect of bacterial metabolism in the modulation of chemotherapeutic response and efficacy in C. elegans. The interaction with E. coli and/or Comamonas aquatica, led to decreased anticolorectal cancer drugs (5-fluorouracil and 5-fluoro-2-deoxyuridine) efficacy and showed cytotoxic effect through bacterial ribonucleotide metabolism in C. elegans. The host-microbe interaction in C. elegans model is a powerful tool in providing insight into different bacteria, its

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products and functions which is amenable to high throughput genetic screening for modulating

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various molecular mechanisms, disease pathology/pathogenicity and drug development.

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Bacterial pathogenicity

Many research reports showed that various bacterial species are pathogenic to C. elegans during

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the search for bacterial food in various environmental compartments (Couillault and Ewbank,

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2002). Although, in the natural habitat the impact of other microorganisms such as fungi and

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viruses have also been studied, but reports are very few as summarized recently in a review article by Jiang and Wang (2018). Thousands of bacterial mutants such as Pseudomonas aeruginosa (Tan

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et al., 1999; Gallagher and Manoil, 2001), Burkholderia pseudomallei (Gan et al., 2002), Serratia marcescens (Kurz et al., 2003), Staphylococcus aureus (Bae et al., 2004), and other pathogenic

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microbes have been tested with C. elegans. Bacterial pathogenicity towards C. elegans is of different categories which vary in different bacterial species. One of the categories involves the slow/prolonged killing of C. elegans by P. aeruginosa as a result of microbial infection and colonization of live microbe in the intestines. Another category involves a rapid/fast killing of C. 7

elegans by P. aeruginosa grown in high osmolarity environment mediated by the production of diffusible toxin (Mahajan-Miklos et al., 1999; Tan et al., 1999). Similarly, a red death mechanism by P. aeruginosa PA01 upon exposure to heat stress and nutrient depletion leading to the

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production of pyoverdin and presence of a reddish complex in the worm’s intestine and pharynx as explained by Zaborin et al. (2009). Kirienko et al. (2013) also identified a liquid-killing model by P. aeruginosa PA01 which results in induction of hypoxia and death in C. elegans through phosphatase activity of histidine kinase B and siderophores pyoverdin as iron chelator. A slower killing mechanism by Salmonella typhimirium is seen in C. elegans resulting into proliferation and

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persistent infection in the worm’s intestine thereby causing its death in few days (Aballay et al.,

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2000). Darby and colleagues (1999) described a killing model by mutant strains of P. aeruginosa

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PA01 against C. elegans through the production of diffusible factors regulated by quorum sensing

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proteins (LasR/RhIR) resulting to hypercontracted muscle and paralysis. Table 1 shows the list of

different mechanisms.

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pathogenic bacteria and their metabolites known to exhibit toxic properties of C. elegans via

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Some bacterial species enter and colonize the gut of C. elegans through different routes viz; oral,

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anal and cuticle, form biofilm and the action of virulence factors lead to slow killing of the worm. Yersinia pestis and Y. pseudotuberculosis infect C. elegans through the attachment and formation

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of biofilm in its digestive tract and cuticle (particularly on the head) (Tan and Darby, 2004). This causes blockage in its feeding, colonization of pathogen in the worm’s intestine as well as biofilm

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independent mechanism through expressed TcaA encoding insecticidal toxin leading to gut distention (Darby et al., 2002; Joshua et al., 2003; Styer et al., 2005; Spanier et al., 2010). Garsin et al. (2001) reported slow killing of C. elegans by Enterococcus faecalis after intestinal colonization, however, the same strain with a deletion of quorum sensing gene such as fsrB results 8

in loss of killing properties, but possesses same colonizing efficiency as compared to the wild strain. Similarly, Burkholderia cenocepacia kills C. elegans gradually when both were grown on standard media showing extensive colonization of the intestine and death of the worm within 1-4

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days through cep quorum-sensing system (Kothe et al., 2003). Serratia marcescens is a broadhost-range, opportunistic pathogen that colonizes C. elegans intestine with median lethal time (LT50) resulting in its death within 6-7 days (Kurz et al., 2003). Staphylococcus aureus and S. epidermidis infect and kill C. elegans, the latter organism does that slowly after colonization, exopolysaccharide synthesis by intercellular adhesion locus (ica) genes and formation of biofilm

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(Begun et al., 2007). Similarly, some bacterial species use extracellular serine protease as virulence

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factor which involves its slow killing after intestinal colonization (Sifri et al., 2002; Geng et al.,

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2016; Ju et al., 2016). The pathogenic effect of Microbacterium nematophilum on C. elegans was

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discovered through contamination of bacteria in a laboratory culture of C. elegans where it colonizes the nematode’s rectum and perianal region thereby resulting in stunted growth,

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constipation and swelling (Hodgkin et al., 2000; Parsons and Cipollo, 2014).

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There are different bacterial genera that use different mechanisms for effective rapid killing of C.

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elegans (Table 1). Bacillus thuringiensis produces a pore-forming crystal toxin, Cry5B that causes intestinal damage to C. elegans in a manner as observed in toxin-treated insects (Marroquin et al.,

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2000). C. elegans are rapidly killed by some species of Streptococci such as S. pneumonia, S. pyogenes and S. agalactiae through the synthesis of hydrogen peroxide in sufficient amount

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(Garsin et al., 2001; Jansen et al., 2002; Bolm et al., 2004). Depending on the strain and culture conditions, the rapid killing of C. elegans by Pseudomonas aeruginosa can be characterized by diffusible toxin such as pyocyanin, a pigmented secondary metabolite which has been identified as a redox-active compound in the presence of high osmolarity medium (Mahajan-Miklos et al., 9

1999). Also through the production of cyanide in rich medium which ceases pharyngeal pumping/blocks respiratory electron transport chain followed by progressive paralysis leading to

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the death of C. elegans within 2hr (Darby et al., 1999; Tan et al., 1999).

Mechanisms of resistance and longevity against bacterial infection

Many reports have identified bacterial pathogenesis carried out by fast killing due to toxic products or slow killing as a result of bacterial colonization in C. elegans intestine (Tan et al., 1999; Cezairliyan et al., 2013; Dzvova et al., 2017). It is also reported that bacterial infections such as

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Photorhabdus luminescens impede the egg laying by hatching of the egg inside C. elegans leading

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to its death (Tan and Shapira, 2011). The details about the bacterial infection of C. elegans and its

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genes/ key molecules are well explained in various articles (Alegado et al., 2003; Durai et al.,

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2011; Balla and Troemel, 2013; Kirienko et al., 2014; Chen et al., 2017; Sharika et al., 2018). With continuous exposure to pathogenic bacteria such as Salmonella enterica, Serratia marcescens,

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Bacillus thuringiensis and Pseudomonas aeruginosa, C. elegans has evolved by developing

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different resistance mechanisms such as aversive learning response through altered feeding preference of non-pathogenic bacteria such as Photorhabdus luminescens, Lactobacillus

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acidophilus, E. coli OP50, and HB101, behavioral and innate immune response as well as

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oxidative stress in order to protect themselves (Zhang et al., 2005; Tenor and Aballay, 2008; Ermolaeva and Schumacher, 2014). The resistance mechanisms in C. elegans against pathogenic

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bacteria such as olfactory avoiding behavior, innate immune system and oxidative stress response have been elucidated schematically in Figure-2. A recent report showed that not only the live bacteria, but also dead bacteria of the species Enterococcus such as E. faecalis and E. faecium induce a rapid and similar host defense responses in C. elegans via immune and stress signaling 10

pathways (Yuen and Ausubel, 2018). The chemotactic property of C. elegans is a behavioural response involved in both searching of food material and protection against infection by pathogenic bacteria. C. elegans can discriminate between pathogenic versus non-pathogenic

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bacteria via an olfactory mechanism for sensing secondary metabolites/products produced by bacteria (Meisel et al., 2014). It can modify its olfactory preferences after exposure to the pathogenic bacteria, learns to avoid the pathogens and thereby increases its attractive power towards odours produced by non-pathogenic bacteria (Zhang et al., 2005). C. elegans recognizes bacterial food or discriminate pathogens with the help of olfactory sensory neurons which is

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enabled by the chemicals that may be either secondary volatile metabolites or quorum sensing

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(QS) signaling molecules produced by bacteria (pathogenic and non-pathogenic) (Meisel et al.,

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2014; Xu et al., 2015). There are few reports on chemotactic property of C. elegans towards the

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chemical produced by bacteria such as acylated homoserine-lactone, a QS signaling molecules produced by Pseudomonas aeruginosa PAO1 (Beale et al., 2006). Similarly, indole produced by

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many bacteria is also considered as one of the QS signaling molecules, which involved in

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interspecies and inter-kingdom level communication in the natural environment (Lee et al., 2015c).

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A recent report discussed on the significant role indole and indole producing bacteria play in attracting C. elegans as opposed the repellent properties of non-indole producing bacteria (Lee et

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al., 2017). Lee et al. (2017) also reported that the indole producing as well as non-indole producing bacteria showed varying effects on the egg-laying behavior of C. elegans. A recent report identified

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the role of Toll-like receptor signaling which activates the BAG (a gas sensing neuron in C. elegans with bag-like ciliary morphology) (Ward et al., 1975; Perkins et al., 1986; Bretscher et al., 2011) and results in pathogen avoidance behaviour (Sowa et al., 2015). Similarly, AWCON (amphid wing

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“C”cells) an amphid sensory neuron in C. elegans helps in the recognition of bacterial QS signaling molecules (Werner et al., 2014). Bacterial toxic metabolites such as tambjamine YP1 and violacein synthesized by E. coli clone

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AA11 showed innate avoiding behaviour and aversive olfactory learning behaviour in C. elegans, respectively (Ballestriero et al., 2016). Meisel et al. (2014) describe that the secondary metabolites such as phenazine-1-carboxamide and pyochelin produced by Pseudomonas aeruginosa PA14 act as environmental cues for recognition and help to avoid pathogenic bacteria. Serrawettin W2, a cyclic lipopentapeptide characterized as biosurfactant produced by Serratia marcescens showed

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repulsive properties to C. elegans (Pradel et al., 2007). Apart from the olfactory learning

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behavioural properties, C. elegans protects itself from pathogenic bacteria through the innate

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immune system (Nicholas and Hodgkin, 2004b; Ermolaeva and Schumacher, 2014). The detailed

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cascade pathways involved in innate immune and stress responses in C. elegans against the microbial infection have been well characterized by molecular and biochemical approaches

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(Ermolaeva and Schumacher, 2014). Although a recent review summarizes that the lifespan of C.

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elegans is regulated by a diverse range of gene products (Uno and Nishida, 2016), however, only

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the regulation of genes stimulated by bacteria or bacterial products was focused. Several reports show that three major signaling pathways such as transforming growth factor-beta (TGF-β)

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pathway, p38 mitogen-activated protein kinase (p38MAPK) pathway, and DAF-2/DAF-16 signaling pathway are involved in the development of innate immunity in C. elegans against

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pathogenic bacteria, homeostasis, and adaptation to specialized stress responses thereby controlling its lifespan (Millet and Ewbank, 2004; Zugasti and Ewbank, 2009; Engelmann and Pujol, 2010; Battisti et al., 2017; Lee and Mylonakis, 2017; Xiao et al., 2017). An excellent review

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by Ermolaeva and Schumacher (2014) discussed in detail the involvement of multiple signaling cascades activated upon exposure of C. elegans to pathogens. The transforming growth factor β (TGF-β)/DBL-1 pathway activate some target genes which

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provide defence against the microbial infection (Mochii et al., 1999; Mallo et al., 2002; Engelmann and Pujol, 2010). In case of mitogen-activated protein kinase (MAPK) pathway, three cascades in C. elegans; p38 MAPK, extracellular signal-regulated kinase (ERK) pathway and kinase protein MEK-1 of the c-Jun N-terminal kinase (JNK) pathway are involved in providing immunity against infections by fungi, Gram-negative and Gram-positive pathogenic bacteria thereby controlling the

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worm’s longevity (Kim et al., 2002; Huffman et al., 2004; Kim et al., 2004; Nicholas and Hodgkin,

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2004a; Engelmann and Pujol, 2010; Xiao et al., 2017). In C. elegans, the transcriptional analysis

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showed that DAF-2 work in a similar way as insulin signaling pathway, in regulating innate

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immunity and resistance to abiotic stresses and controlling lifespan (Evans et al., 2008; Engelmann and Pujol, 2010). DAF-2 is a homolog of insulin/insulin-like growth factor-1 receptor, which

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regulates a FOXO family transcription factor named DAF-16. DAF-2 and daf-2 mutant DAF-16

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are involved in either activating or repressing stress response, detoxification, metabolism,

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autophagy, and antimicrobial effectors molecules related genes (Evans et al., 2008; Engelmann and Pujol, 2010; Tsuchiya et al., 2013; Webb and Brunet, 2014; Uno and Nishida, 2016). The

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antimicrobial effectors molecules directly or indirectly regulated by DAF-2 and DAF-16 include lysozyme (Lys-7 gene) (Mallo et al., 2002; O'Rourke et al., 2006), saposin (spp-1 gene) (Kato et

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al., 2002), glycine/tyrosine-rich antimicrobial peptides (NLP-31 gene) and hypothetical protein Y43C5A.3 (Lim et al., 2016), Ascaris suum antibacterial factor (ASABF)-type antimicrobial peptide (abf-2 gene) (Kato et al., 2002), and lectin (O'Rourke et al., 2006; Ideo et al., 2009). The functional genes of each effector molecule are regulated by DAF-2 and DAF-16 via the help of 13

certain cascades of kinase proteins. Several recent and past studies on transcriptional profiling of the above kinases confirmed the diverse physiological role in the life cycle of C. elegans. In spite of resistance development against pathogenic bacteria, C. elegans uses different signaling

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mechanisms against some bacteria, which increase the lifespan and thereby minimize ageing. The well-characterized molecular signaling mechanism involved in the extension of lifespan of C. elegans after interaction with bacteria and bacterial products includes activation of different cascade protein as explained in Figure 1.

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Bacteria extending the lifespan of C. elegans

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In natural habitats, C. elegans are encountered by complex microorganisms including both

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pathogenic and non-pathogenic bacteria (Felix and Braendle, 2010; Dirksen et al., 2016; Samuel

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et al., 2016). Many research reports and review articles are available on the topic of neuronal behaviour connection with the functionality in different species including the human and C.

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elegans (Bishop and Guarente, 2007; Aballay, 2009; Clark and Hodgkin, 2013; Berg et al., 2016).

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Little is known about the composition of naturally occurring gut flora in C. elegans, but there is

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evidence on the commensal and mutualistic interaction among nematodes and microorganisms as explained in a review by Midha et al. (2017). With the aim of enhancing the lifespan of C. elegans,

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diverse bacteria have been isolated and characterized from different environmental habitat, which may also serve as food material for the nematode (Lee et al., 2015b; Park et al., 2015; Nakagawa

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et al., 2016; Zhao et al., 2017). Although in the laboratory, E. coli OP50 is used as food for C. elegans (Samuel et al., 2016), many bacterial species such as Gluconobacter sp., Providencia sp., Corynebacterium glutamicum, Enterobacter sp., Bacillus subtilis, B.megaterium, B. firmus, Pseudomonas fluorescens, and Micrococcus luteus characterized from different environments 14

serve as good food sources compared to the E. coli OP50 (Abada et al., 2009; Coolon et al., 2009; Gusarov et al., 2013; Samuel et al., 2016; Liu et al., 2017). There is much ongoing research for the analysis of microbiome for both, C. elegans gut and natural habitat to discuss the importance of

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the microbes in the life cycle of nematodes (Felix and Duveau, 2012; Dirksen et al., 2016; Samuel et al., 2016). It is noteworthy that the role of bacteria in extending the lifespan of C. elegans can be determined by monitoring the accumulation of biochemical marker protein of ageing such as carbonyl and lipofuscin (Komura et al., 2013). A recent report showed that the analysis of proteins of microbiome members as compared to the laboratory food bacterium by proteomic approach will

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help to identify the molecular process happening during the time of host-microbe interactions

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(Cassidy et al., 2018). Reports show that the oral administration of lactic acid bacteria (LAB)

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improves the balance of intestinal microbial community thus protecting it from the microbial

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infection (Ikeda et al., 2007; Komura et al., 2012). Same ways as probiotics play beneficial role(s) in humans; they also colonize in the intestine of C. elegans thus minimizing the chances of

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pathogenic bacteria colonization. Several newly identified LAB from different sources showed

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enhanced longevity of C. elegans (Table 2). It is reported that the inhibitory properties of LAB

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against pathogenic bacteria are through secretion of antimicrobial compounds such as organic acids, hydrogen peroxide and bacteriocins (Westbroek et al., 2010). Many reports showed that the

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feeding of C. elegans with probiotic bacteria either singly or mixed with standard food E. coli OP50 results in an increased lifespan (Ikeda et al., 2007; Komura et al., 2013; Zhao et al., 2013).

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The different signaling pathways or molecular mechanisms involved in increasing the lifespan of C. elegans by LAB have been described in several studies (Komura et al., 2013; Lee et al., 2015a; Kwon et al., 2016). Komura et al. (2012), experimentally proved that feeding of nematode with Bifidobacteria results in the development of defence mechanism against Legionella. Feeding of C. 15

elegans with heat-killed Lactobacillus plantarum 133 and L. fermentum 21 results in enhanced survivability by protecting it against Salmonella and Yersinia infection (Lee et al., 2015b). Weissella species isolated from fermented kimchi showed increased lifespan of C. elegans by

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activating DAF-16 via the c-Jun-N terminal kinase pathway (Lee et al., 2015a). Furthermore, Komura et al. (Komura et al., 2013) also reported that the cell wall component of Bifidobacteria involves in the increased lifespan of C. elegans via the activation of skn-1 gene which is regulated by p38 MAPK pathway.

Currently, microbe-mediated protection is an emerging trend on the topic of host-microbe

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interaction, which down-regulates the virulence factor of powerful pathogenic bacteria, thereby

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minimizing the infection of the host. A probiotic Enterococcus faecalis Symbiofloar when fed

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along with E. coli O157: H7 resulted in the down-regulation of virulent genes such as locus for

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enterocyte effacement (LEE), flagellum and quorum sensing in E. coli O157: H7 pathogen thereby

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increasing the lifespan of C. elegans (Neuhaus et al., 2016). Colonization of mildly pathogenic

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Enterococcus faecalis to the worm intestine also provides protection against pathogenic bacteria such as Staphylococcus aureus by production of antimicrobial superoxide (King et al., 2016). A

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recent report showed the ability of Propionibacterium freudenreichii, non-lactic acid probiotic bacteria, to extend the lifespan of C. elegans by the binding of a TGF-β-like ligand to TGF-β

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receptors and activation of PMK-1 and TGF-β target genes via p38 MAPK and TGF-β pathways

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involved in innate immunity (Kwon et al., 2016). Few reports showed that nitric oxide produced by Bacillus subtilis enhance the longevity of C. elegans (Gusarov et al., 2013). The molecular mechanism for increasing the longevity as well as stress resistance by nitric oxide is via a set of genes, regulated by HSF-1 and DAF-16 transcription factors. Autophagy is another way of extending the lifespan of C. elegans, induced by numerous 16

factors including the bacteria. Abada et al. (2009) reported for the first time the role of autophagy activation in the extension of C. elegans lifespan through feeding on Bacillus mycoides and B. soli. Furthermore, Streptomyces venezuelae induced autophagy in C. elegans, which increases its

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lifespan by protecting it from Pseudomonas aeruginosa infection (Zou et al., 2014). Although, the chemical structure of the metabolite (MW <300) isolated from Streptomyces venezuelae has not been characterized but its role in autophagy through induction of glutathione proteasomal damage, proteasomal disturbance and PINK1 dependent autophagy has been described (Martinez et al., 2015). The molecular signaling pathway involved in the extension of the lifespan of C. elegans

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after interaction with different species of bacteria is explained in Figure 1.

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As a result of recent advances in the research technology, there are several reports on the

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application of different approaches for the quenching of QS molecules/circuit in pathogenic

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bacteria which protects or prolong the nematode’s lifespan (Guo et al., 2013; Hirakawa and Tomita, 2013). However, our focus is to discuss several bacteria producing compounds that

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interfere with QS circuits. Thus, the pathogenic bacteria ingested by C. elegans can be eradicated

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by treating with the quorum quenching molecules.

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The enzymatic degradation properties of signaling molecules have been reported in many bacteria (Park et al., 2003; Park et al., 2006; Mei et al., 2010; Mayer et al., 2015; Tang et al., 2015).

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Secretion of acyl homoserine lactone (AHL) acylase encoded by aiiD gene from Ralstonia sp. quenched quorum sensing, decreased motility and virulence factor production in P. aeruginosa

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when tested on C. elegans (Lin et al., 2003). In a study by Koch and colleagues (Koch et al., 2014), Deinococcus radiodurans secreted two potent quorum quenching enzymes- AHL lactonase (QqlR) and AHL acylase (QqaR) with degradative potential against long acryl chain AHL without 3-oxo substitution. The catalytic activity led to the down regulation of virulence genes (LasB encoding 17

for protease elastase) in bacteria thus, increasing C. elegans lifespan. Similarly, AHL lactonase (MomL) found in Muricauda olearia strain Th120 shows degradative activity against short and long chain AHLs with or without substitution of oxo-group at the C-3 position (Tang et al., 2015).

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Thus, this enzymatic degradation results in the protection of C. elegans from death via attenuation of P. aeruginosa virulence.

A recent report identified the role of intestinal Enterococcus faecium secreting antigen A (SagA) which possesses peptidoglycan hydrolase activity that involved in the protection of C. elegans against Salmonella pathogenesis (Rangan et al., 2016).

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Thus, effective quorum quenching enzymes produced by bacteria are potential competitive

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strategy in attenuating or down regulating virulence gene expression and protecting C. elegans

Conclusions and future perspective

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from pathogenic bacterial killing which could serve as therapeutic agents.

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The bacteriovorus C. elegans is present in different environmental habitats, where chances of its

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frequent exposure to different bacterial genera and species occur. The diversity of bacteria (i.e.

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pathogenic and non-pathogenic) in the natural habitat of C. elegans opens new doors to study several molecular and biochemical phenomena related to host-microbe interaction. There are

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increasing reports on the topic of worm-microbe interaction for the study of immunological responses, antimicrobial properties against microbes and killing of the worm either by secondary

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metabolites or by colonizing the intestine. The interaction between bacteria or bacterial products and C. elegans in the environment involved in stimulating positive or negative effects on the lifespan in multiple ways are represented schematically in Figure-3. The applications of C. elegans as a model organism for the study of microbial pathogenesis are still new and the extent of their 18

relevance to human disease remains yet to be determined. The virulence factors involved in the pathogenesis of foodborne pathogenic bacteria can be screened in-vivo using C. elegans to reduce the concern of resistance to multiple antibiotics (Aksoy and Sen, 2015). After in-vivo screening

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and identification of virulence factors in C. elegans, the detailed characterization of these factors in another model organism such as mouse is required.

The composition of C. elegans gut microbiota is greatly influenced by its developmental stage and genotype which is totally independent of the microbial composition in its natural habitat (Dirksen et al., 2016). Further research is required to study the complex gut microbial community and how

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microbiota interacts with C. elegans in their natural environment. Apart from the longevity of C.

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elegans, some bacterial products have been characterized as chemo-attractants which help C.

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elegans to discriminate between pathogenic and non-pathogenic bacteria. The signaling

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mechanism(s) involved in the chemotactic response to the odorant compounds released by bacteria needs to be addressed. There are several bacterial products identified as biofilm inhibitors which

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interfere with the quorum sensing circuits occurring among pathogenic bacteria and may indirectly

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enhance the lifespan of C. elegans by rescuing it from infection as schematically depicted in Figure

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3.

Furthermore, these potential inhibitors need to undergo clinical trial in animals including humans.

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Though there are several species of bacteria which have been shown to affect C. elegans longevity, the mechanisms are unknown. To escape from pathogenic bacteria, C. elegans triggers protective

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mechanisms such as olfactory avoidance learning behaviour by sensing microbial products as reported in Table 1, however, if C. elegans gets infected with pathogen via ingestion, it activates its innate immunological responses (Ermolaeva and Schumacher, 2014). The production of antimicrobial peptides by C. elegans is an antibacterial defence (Ewbank and Zugasti, 2011). C. 19

elegans can be used to screen novel and potent immunological effectors molecules as potential antimicrobial drugs against several pathogenic bacteria (Artal-Sanz et al., 2006; Ewbank and Zugasti, 2011; Squiban and Kurz, 2011; Arvanitis et al., 2013; Ermolaeva and Schumacher, 2014).

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Some of the LAB showed enhanced longevity in C. elegans by activating innate immunity pathways, however, the key molecules from LAB involved in longevity need to be identified and characterized. The biofilm inhibitory molecules involved in rescuing C. elegans from pathogenic bacteria can be applied for the control of pathogenesis in other species including human. Molecules produced by beneficial bacteria residing in the gut of C. elegans can be further biochemically

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characterized and elucidating the molecular mechanism of defence would be of great interest.

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Since intestinal surface would be the first line of entry and attachment by microbe, the

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identification of surface recognition pattern in bacterial cell membrane and C. elegans intestinal

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wall surface would be a promising future research plan. With the help of olfactory learning properties of C. elegans, future work is needed to explore more bacterial QS signaling molecules

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which would be helpful in identifying quorum quenching drug that may help in treating infections.

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The bacterial products which showed nematicidal effect can also be used as target molecule(s) for

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designing and development of therapeutic drug(s).

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Declaration of interest

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The authors report no declarations of interest.

Acknowledgement Authors acknowledge the School of Engineering and Technology, Department of Biotechnology, Sharda University, India for her financial support. 20

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Table 1 List of bacteria and bacterial metabolites known to exhibit toxicity properties of C. elegans

Pseudomonas brassicacearum strain DF41 B. thuringiensis

Bacterial products/enzymes

Properties (mode of action)

References

Serine protease

Biocontrol activity against C. elegans

(Geng et al., 2016) (Nandi et al., 2016)

Lipopeptide sclerosin, hydrogen cyanide and degradative enzyme

A

Bacteria Diffusible toxin production B. firmus DS-1

Crystal protein Cry6Aa

Photorhabdus luminescens

and

M

Bacillus cereus

Pyrrolnitrin cyanide Cry B5 toxin

hydrogen

ED

P. chlororaphis strain PA23

Cause C. elegans death by toxic metabolites and biofilm formation on the head of worm The crystal protein showed toxicity to C. elegans through necrosis signaling pathway resulting in lysosomal lysis Exhibit both nematicidal and repellent activity Virulence to C. elegans through toxin production

Toxin complex (Tc) Makes caterpillars floppy (McF) toxin 1-hydroxyphenazine Phenazine-1-carboxylic acid Pyocyanin

Vulnerability in intestinal cells of C. elegans, development of crystal-like toxin within the intestinal lumen and depletion of pmk-1 by RNAi leading to damage of the midgut and deformation Rapid death of C. elegans occurred at wide range of pH and different environmental factors through shedding of the worm’s cuticle

Metabolites such as bacteriocin

Reduction in growth and development of C. elegans Hydrogen cyanide performed as chemotactic repellent to nematode and combined effect of hydrogen cyanide and 2,4-DAPG result in death of nematode

Yersinia enterocolitica

Hydrogen cyanide and 2,4diacetylphloroglucinol (2,4DAPG) tcaA gene

Nontyphoidal salmonellae

Toxin (cytotoxin and endotoxin)

Death occurs through gastroenteritis

P. aeruginosa (strain PA14 and PAO1)

Hydrogen cyanide

Death through inactivation of hcnC gene which encodes for the hydrogen cyanide synthase

PT

Pseudomonas aeruginosa

CC E

Lactobacillus reuteri and Pediococcus acidilactici

A

P. fluorescens CHA0

Encodes an insecticidal toxin in the nematicidal activity against C. elegans

Colonization and infection

31

(Zhang et al., 2016) (Nandi et al., 2015) (Iatsenko et al., 2014b) (Sato et al., 2014) (Cezairliyan et al., 2013)

(Fasseas et al., 2013) (Neidig et al., 2011) (Spanier et al., 2010) (Jesudason et al., 2006) (Gallagher and Manoil, 2001; Wareham et al., 2005)

N U SC RI PT

Pseudomonas syringae MB03

pepP, clpA and clpS

Bacillus nematocida B16

Cronobacter sakazakii

Proteases with broad ranges of substrates Vibrio cholerae cytolysin (VCC) Invasion protein antigen-lpa proteins (lpaB, C and D) Exopolysaccharide Matrix Lipopolysaccharide (LPS)

Leucobacter chromiireducens

Biofilm formation

Microbacterium nematophilum Listeria monocytogenes

Deformed anal region (Dar) phenotype ActA, PrfA and DegU

Colonizes the reproductive tract through the external vulva opening and initiates a lethal uterine infection Colonizes the rectum of infected worm and results in localized swelling, coprostasis, and retardant growth Infects worm and accumulates in its intestine causing death

Virulence gene

Gut infection

Vibrio cholera Shigella flexneri

M

ED

PT

Staphylococcus aureus

A

Pseudomonas aeruginosa

Transcriptional analysis showed that the nematicidal genes present in Pseudomonas strain get up-regulated upon the infection under different nutritional condition Killing of C. elegans through intestinal colonization by trojan horse mechanism Infection and killing of C. elegans as a result of induction of wide variety of immune responses The genes are kinetically regulated in C. elegans during infection Inhibiting specific host responses to microbes and induction of NPR-1-neuropeptide receptor dependent behaviours of C. elegans Accumulates inside the host and induce antimicrobial genes in C. elegans

Virulence factor

Gut infection and cell death

Microbacterium nematophilum

Extracellular signal-regulated kinase and mitogen-activated protein kinase cascade Extracellular toxin

This bacterium adheres to C. elegans rectum and postanal cuticle which causes swelling of hypodermal tissue and constipation

Virulence gene involved in lipopolysaccharide biosynthesis, iorn uptake and hemolysin production Extracellular matrix

Gut infection and tissue degradation

CC E

Salmonella enteric

Burkholderia cenocepacia

A

Serratia marcescens

Yersinia pestis Yersinia pseudotuberculosis Enterococcus faecalis

Virulence is due to protease gene

Gut colonization, infection and death through cep mediated quorum sensing system

Formation of extracellular biofilm results in interference with the feeding of C. elegans Gut infection, colonization and proliferation in adult C. elegans intestine resulting in rapid killing

Other diffusible factors

32

(Ali et al., 2016) (Niu et al., 2012) (Sahu et al., 2012) (Kesika et al., 2011) (Reddy et al., 2011) (Sivamaruthi et al., 2011) (Muir and Tan, 2008) (Akimkina et al., 2006) (Thomsen et al., 2006) (Bae et al., 2004; Begun et al., 2005) (Tenor et al., 2004) (Nicholas and Hodgkin, 2004a) (Kothe et al., 2003) (Kurz et al., 2003)

(Darby et al., 2002) (Sifri et al., 2002)

N U SC RI PT

Alcaligenes faecalis ZD02

Extracellular serine protease

Nematicidal activity against C. elegans

Acinobacter johnsonii MB44

Membrane protein A, phospholipase A and penicillin binding protein Virulence protease

Nematicidal activity on C. elegans due to the presence of potential virulence proteins

Degradation of the chitin on egg or cuticle of the nematode via Enp (extracellular neutral proteases) Nematicidal to C. elegans

(Zheng et al., 2016) (Neu et al., 2014)

Inhibitory effect on C. elegans

(Iatsenko et al., 2014a)

Indole, indole-3-carboxyldehyde and indole-3-acetic acid

The indole and indole derivatives from these pathogenic bacteria act synergistically for killing C. elegans

(Bommarius et al., 2013)

Virulence factors

Nematicidal activity

(Gan et al., 2002; Lee et al., 2011) (Caldwell al., 2009)

M

A

Pentabromopseudilin

Ketones (2-nonanone, 2heptanone, and 2-undecanne) and dimethyl sulfide

CC E

PT

ED

Fictibacillus phosphorivorans G25-29 Pseudoalteromonas luteoviolacea S4060 P. tunicate D2 P. rubra S2471 P. piscicida S2049 Pseudomonas chlororaphis strain 449 Serratia proteamaculans strain 94 Enteropathogenic E. coli, Enterohemorrhagic E. coli and commensal Escherichia coli Burkholderia pseudomallei

Streptomyces venezuelae

A

Salmonella enterica Typhimurium Clostridium spp.

serovar

(Ju et al., 2016) (Tian et al., 2016)

Lipophilic secondary metabolites DNA adenine methyltransferase (DAM) Clostridial collagenase (Type III)

Disruption of the ubiquitin-proteasome system function resulting in gradual degeneration of dopamine neuron (dopaminergic cell death) in C. elegans Methylation by DAM involved in modulation of C. elegans virulence Degrades the supports between the nematode’s inner and outer cortical layers

33

(Oza et al., 2005) (Cox et al., 1981)

et

N U SC RI PT

Table 2 List of bacterial species known to enhance the longevity of C. elegans Properties (mode of action)

Ref

L. rhamnosus R4

Extend the nematode’s lifespan via antioxidant and antimicrobial activity of the LAB

Enterococcus faecalis

Colonizes the intestine and defends C. elegans against infection of potent virulent Staphylococcus aureus via the production of antimicrobial superoxide

Enterococcus faecalis Symbioflor ® E. coli O157: H7

Down regulation of LEE, flagellum and quorum sensing genes of pathogens in C. elegans

Lactobacillus spp.

Heat-killed Lactobacillus spp. protect and enhance the survivability of C. elegans against Salmonella and Yersinia infection

(Az 201 (Kin 201 (Ne al., 2 (Lee 201 (Phu al., 2 (Zha 201 (Iats al., 2 (Yu 201 (Gu al., 2 (Ko al., 2 (Mo Katz 201 (Mo Katz 201

M

PT

B. subtilis GS67

ED

Bacopa monnieri (L.) Lactic acid bacteria

CC E

B. licheniformis

A

Bacteria

Acts as potent reactive oxygen species scavenger which enhances the survival of the worms in thermal and oxidative stress conditions Protect C. elegans from toxic effect of grapheme dioxide by maintaining the normal intestinal permeability Protects C. elegans from Gram-positive pathogen via fengycin-mediated microbial antagonism Protects the C. elegans from the infection caused by Staphylococcus aureus Nitric oxide (NO) produced by B. subtilis facilitates increased longevity and heat shock response in C. elegans

Bifidobacteria

Cell wall component of Bifidobacteria increases the lifespan of C. elegans via the activation of skn-1 gene which is regulated by p38 MAPK pathway

B. megaterium

Enhances resistance in C. elegans against pathogens, although through different mechanisms, this strain led to increased resistance as a secondary consequence of compromised reproduction

A

B. subtilis

Pseudomonas mendocina

Protects the worm by enhancing its resistance to pathogens through p38 MAPK-dependent priming of the immune system

34

N U SC RI PT

Lactobacilllus and Bifidobacterium

Promote the life span of the nematode through p38 MAPK pathway, enhance host defence mechanisms and confer resistance against pathogenic bacteria

L. rhamnosus CNCM I-3690

A

E. coli GD1

ED

Escherichia coli NM6003

CC E

PT

E. coli AroD

E. coli HT115 B. mycoides B. soli

Bacillus megaterium

A

M

L. acidophilus

B. infantis

(Ike al., 2 Kom al., 2 Lengthens the worm's life expectancy and induces different expression of DAF-16/insulin-like pathway (Gro et al Dose-dependent lifespan extension relative to E. coli OP50 (Go al., 2 Protective effect mediated by p38 MAPK and β-catenin signaling pathways (Kim Myl 201 C. elegans with a gene (rde-2) contributing in transposon silencing and RNA interference showed an increased (Liu lifespan when fed with DsrA deletion mutant E.coli NM6003. This could be attributed to the increased 201 expression level of diacylglycerol lipase gene, F42G9.6 and the absence of DsrA which blocks or suppresses daf-2 expression Lifespan extension relative to HT115 (Vir 201 Mechanism is likely via activation of host protective pathways rather than the production of bacterial (Ko metabolites or gut colonization al., 2 Worms fed with E. coli HT115 expressing RNA interference showed altered metabolic profile, increased ability (Rei to sense more signalling amino acids and extended lifespan when compared with E. coli OP50 al., 2 Extend the life span of C. elegans by activating the autophagic process (Ab al., 2 Increase the worm’s lifespan via defence genes up regulation (Co al., 2

35

Figure legends Figure 1. Schematic representation of different signaling pathway triggered by diverse bacteria involved in the extension of lifespan of C. elegans (information from literature by Clark and

A

CC

EP

TE

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Hodgkin, 2013; Komura et al., 2013; Lee et al., 2015a; Martinez et al., 2015; Kwon et al., 2016).

Figure 2. Two major resistance mechanisms in C. elegans against pathogenic bacteria such as 1olfactory avoiding behaviour and 2-innate immune system and oxidative stress response (through

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TGF-β, JNK/MAPK, and AMPK signalling pathways) (information from literature by Ermolaeva

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and Schumacher, 2014; Meisel et al., 2014; Park et al., 2017).

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Figure 3. Diagrammatic explanation about the consequences of interaction between bacteria or bacterial products with C. elegans. A: Diverse of bacteria in the natural habitat attract C. elegans

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chemotactively for food search; B: Formation of biofilm by pathogenic bacteria inside the C.

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elegans; C: Signaling molecules from non-pathogenic chemotactively attracts C. elegans and from pathogenic bacteria helps in olfactory aversive learning; D: Quorum sensing molecules secreted

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by pathogenic bacteria responsible for the pathogenesis; E: Different strategy for the inhibiting the quorum sensing circuits; F: Feeding with lactic acid bacteria, non-pathogenic and mild pathogenic bacteria provide protection against the pathogenic bacteria; G: the molecular signaling mechanism/pathways activated by different bacteria involved in the extension C. elegans lifespan; 37

H: Homeostasis of gut microbiota; I: Antimicrobial defense by secreting antimicrobial peptides (AMPs) and proteins; J and K: Slow and fast killing of C. elegans by pathogenic bacteria either

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colonizing in the intestine or by secreting secondary metabolites as toxin.

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