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Listeria monocytogenes is a Gram-positive, intracellular pathogen responsible for the highly fatal foodborne illness listeriosis. Establishing intracellular infections requires the coordinated expressions of a variety of virulence factors, such as the pore-forming toxin listeriolysin O (LLO), in response to various intra- and extracellular signals. For example, we previously reported that L. monocytogenes differentially modulated LLO production in response to exogenous propionate, a short chain fatty acid either used in salt form as a human food ingredient or produced endogenously by gut microbial fermentation. Therefore, propionate is likely a continuously present signal throughout the L. monocytogenes transmission and infection process. However, little is known about the role of propionate in modulating L. monocytogenes-host interactions. Here we investigated the impact of propionate treatment on L. monocytogenes intracellular infections using cell culture infection models. Propionate treatment was performed separately on L. monocytogenes or host cells before or during infections to better distinguish pathogen-versus-host responses to propionate. Intracellular CFU in RAW264.7 macrophages and plaque diameters in L-fibroblasts were measured as proxy for intracellular infection outcomes. Nitrite levels and cellular morphology were also measured to assess host responses to propionate. We found that propionate pretreatment of anaerobic, but not aerobic, L. monocytogenes significantly enhanced subsequent intracellular infections in both cell types and nitrite production by infected macrophages. Propionate treatment of uninfected macrophages significantly altered cell morphology, seen by longer cells and greater migration, and reduced nitrite concentration in activated macrophages. Treatment of macrophages with propionate prior to or during infections significantly inhibited intracellular growth of L. monocytogenes, including those pre-treated with propionate. These results showcased an opposing effect of propionate on L. monocytogenes intracellular infections and strongly support propionate as an important signaling molecule for both the pathogen and the host cell that can potentially alter the outcome of L. monocytogenes-host interactions.

Gold nanoparticles (AuNPs) bound with biomolecules have emerged as suitable biosensors exploiting unique surface chemistries and optical properties. Many efforts have focused on antibody bioconjugation to AuNPs resulting in a sensitive bioconjugate to detect specific types of bacteria. Unfortunately, bacteria thrive under various harsh environments, and an understanding of bioconjugate stability is needed. Here, we show a method for optimizing Listeria monocytogenes polyclonal antibodies bioconjugation mechanisms to AuNPs via covalent binding at different pH values, from 2 to 11, and 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid, NaOH, HCl conditions. By fitting Lorentz curves to the amide I and II regions, we analyze the stability of the antibody secondary structure. This shows an increase in the apparent breakdown of the antibody secondary structure during bioconjugation as pH decreases from 7.9 to 2. We find variable adsorption efficiency, measured as the percentage of antibody adsorbed to the AuNP surface, from 17 to 27% as pH increases from 2 to 6 before decreasing to 8 and 13% at pH 7.9 and 11, respectively. Transmission electron microscopy (TEM) analysis reveals discrepancies between size and morphological changes due to the corona layer assembly from antibody binding to single nanoparticles versus aggregation or cluster self-assembly into large aggregates. The corona layer formation size increases from 3.9 to 5.1 nm from pH 2 to 6, at pH 7.9, there is incomplete corona formation, whereas at pH 11, there is a corona layer formed of 6.4 nm. These results indicate that the covalent binding process was more efficient at lower pH values; however, aggregation and deactivation of the antibodies were observed. We demonstrate that optimum bioconjugation condition was determined at pH 6 and MES buffer-type by indicators of covalent bonding and stability of the antibody secondary structure using Fourier transform-infrared, the morphological characteristics and corona layer formation using TEM, and low wavelength shifts of ultraviolet–visible after bioconjugation.

Listeria monocytogenes is a foodborne pathogen capable of secreting listeriolysin O (LLO), a pore-forming toxin encoded by the hly gene. While the functions of LLO have been studied extensively, how the production of LLO is modulated by the intestinal environment, devoid of oxygen and enriched in short chain fatty acids (SCFAs), is not completely understood. Using L. monocytogenes strain 10403s, we found that hly transcription was moderately decreased by aerobic SCFA exposures but significantly increased by anaerobic SCFA exposures. Moreover, aerobic, but not anaerobic, exposure to low levels of SCFAs resulted in a significantly higher LLO activity. These results demonstrated that transcriptional and post-transcriptional regulations of LLO production were separately modulated by SCFAs and were responsive to oxygen levels. Examining isogenic mutants revealed that PrfA and SigB play a role in regulating LLO production in response to SCFAs. Effects of SCFAs were also present in the cardiotropic strain 07PF0776 but distinctly different from those in strain 10403s. For both strains, prior exposures to SCFAs altered intracellular infections in Caco-2 and RAW264.7 cells and the plaque sizes in L fibroblasts, a result confirming the ability of L. monocytogenes to adapt to SCFAs in ways that impact its subsequent infection outcomes.

Propionate is a common food preservative and one of the major fermentation acids in the intestines. Therefore, exposure to propionate is frequent for foodborne pathogens and likely takes place under suboxic conditions. However, it is not clear whether the absence of oxygen affects how pathogens respond to propionate. Here, we investigated how propionate exposure affects Listeria monocytogenes growth and virulence factor production under aerobic or anaerobic conditions and showed that oxygen indeed plays a key role in modulating L. monocytogenes response to propionate. Under aerobic conditions, propionate supplementations had no effect on planktonic growth but resulted in decreased adherent growth. Under anaerobic conditions, propionate supplementations resulted in a pH-dependent inhibition of planktonic growth and increased adherent growth. Cultures grown with propionate accumulated higher levels of acetoin under aerobic conditions but lower levels of ethanol under both aerobic and anaerobic conditions. Metabolic perturbations by propionate were also evident by the increase in straight chain fatty acids. Finally, propionate supplementations resulted in increased listeriolyin O (LLO) production under anaerobic conditions but decreased LLO production under aerobic conditions. These results demonstrate for the first time that the presence or absence of oxygen plays a critical role in shaping L. monocytogenes responses to propionate.

Fatty acids (FAs) are the major structural component of cellular membranes, which provide a physical and chemical barrier that insulates intracellular reactions from environmental fluctuations. The native composition of membrane FAs establishes the topological and chemical parameters for membrane-associated functions and is therefore modulated diligently by microorganisms especially in response to environmental stresses. However, the consequences of altered FA composition during host-pathogen interactions are poorly understood. The food-borne pathogen Listeria monocytogenes contains mostly saturated branched-chain FAs (BCFAs), which support growth at low pH and low temperature. In this study, we show that anteiso-BCFAs enhance bacterial resistance against phagosomal killing in macrophages. Specifically, BCFAs protect against antimicrobial peptides and peptidoglycan hydrolases, two classes of phagosome antimicrobial defense mechanisms. In addition, the production of the critical virulence factor, listeriolysin O, was compromised by FA modulation, suggesting that FAs play a key role in virulence regulation. In summary, our results emphasize the significance of FA metabolism, not only in bacterial virulence regulation but also in membrane barrier function by providing resistance against host antimicrobial stress.

Whereas cell cycle arrest, apoptosis, and senescence are traditionally thought of as the major functions of the tumor suppressor p53, recent studies revealed two unique functions for this protein: p53 regulates cellular energy metabolism and antioxidant defense mechanisms. Here, we identify glutaminase 2 (GLS2) as a previously uncharacterized p53 target gene to mediate these two functions of the p53 protein. GLS2 encodes a mitochondrial glutaminase catalyzing the hydrolysis of glutamine to glutamate. p53 increases the GLS2 expression under both nonstressed and stressed conditions. GLS2 regulates cellular energy metabolism by increasing production of glutamate and α-ketoglutarate, which in turn results in enhanced mitochondrial respiration and ATP generation. Furthermore, GLS2 regulates antioxidant defense function in cells by increasing reduced glutathione (GSH) levels and decreasing ROS levels, which in turn protects cells from oxidative stress (e.g., H 2 O 2 )-induced apoptosis. Consistent with these functions of GLS2, the activation of p53 increases the levels of glutamate and α-ketoglutarate, mitochondrial respiration rate, and GSH levels and decreases reactive oxygen species (ROS) levels in cells. Furthermore, GLS2 expression is lost or greatly decreased in hepatocellular carcinomas and the overexpression of GLS2 greatly reduced tumor cell colony formation. These results demonstrated that as a unique p53 target gene, GLS2 is a mediator of p53’s role in energy metabolism and antioxidant defense, which can contribute to its role in tumor suppression.

The p53 protein ensures cellular fidelity by suppressing or killing cells under stresses that enhance the mutation rate. Evidence suggests that the p53 protein may also ensure the fidelity of the epigenome. In this study a group of drugs that alter the deoxycytosine methylation patterns in cellular DNA are shown to preferentially kill human and mouse cells that contain p53 mutations or deficiencies. These observations are extended to mice that contain p53 deficiencies or missense mutations in their genome, which are preferentially killed when compared to mice with a wild type p53 gene. This is also the case for human cancer cell xenografts containing p53 mutations, which preferentially are killed by these drugs when compared to similar tumors with wild type p53. The loss of p53 function enhances a synthetic lethality with drugs that block or alter the patterns of deoxycytidine methylation in the genome.

The identification and analysis of the genes encoding perchlorate reductase and chlorite dismutase has provided not only a building block for pathway understanding, but has also provided a tool for bioremediative and phylogenetic studies. On-going genome sequencing will further facilitate transcriptional profiling under perchlorate-reducing conditions via microarray analyses. This analysis will give a more inclusive look into transcriptional expression patterns associated with the perchlorate metabolism. While further advancements in the genetic analysis of perchlorate-reducing bacteria continue, the recent development of a genetic system in D. aromatica will provide an invaluable tool for corroborating microarray results and solidifying hypotheses regarding microbial perchlorate metabolism.

Publisher Summary This chapter examines the diverse geochemical and microbial reactivities of humic substances (HS), the role that these complex organics play in a diversity of environments, and their potential applicability to bioremediative strategies. HS are ubiquitous components in the environment and can be readily isolated from nearly all soils, waters, and sediments. They can account for as much as 10% by weight of the total content of many soils and sediments. It is clear that HS interact with microbial populations through a diversity of mechanisms. These interactions may potentially affect the microbial ecology of a specific environment and significantly affect fate and transport of organic and inorganic environmental pollutants. By considering these interactions, strategies for bioremediation of particular pollutants may be improved. HS can be utilized by microorganisms as effective electron acceptors for the oxidative degradation of organic carbon in anaerobic environments. Alternatively, HS in the reduced form can be utilized by microorganisms as effective electron donors for the assimilation of organic carbon coupled to denitrification. These metabolic processes as well as the intrinsic geochemical reactivity of HS are now known to play an important role on the fate and transport of herbicides, pesticides, hydrocarbons, cations, and other important environmental pollutants.

The response behavior of three dissimilatory perchlorate-reducing bacteria to different electron acceptors (nitrate, chlorate, and perchlorate) was investigated with two different assays. The observed response was species-specific, dependent on the prior growth conditions, and was inhibited by oxygen. We observed attraction toward nitrate when Dechloromonas aromatica strain RCB and Azospira suillum strain PS were grown with nitrate. When D. aromatica and Dechloromonas agitata strain CKB were grown with perchlorate, both responded to nitrate, chlorate, and perchlorate. When A. suillum was grown with perchlorate, the organism responded to chlorate and perchlorate but not nitrate. A gene replacement mutant in the perchlorate reductase subunit (pcrA) of D. aromatica resulted in a loss of the attraction response toward perchlorate but had no impact on the nitrate response. Washed-cell suspension studies revealed that the perchlorate grown cells of D. aromatica reduced both perchlorate and nitrate, while A. suillum cells reduced perchlorate only. Based on these observations, energy taxis was proposed as the underlying mechanism for the responses to (per)chlorate by D. aromatica. To the best of our knowledge, this study represents the first investigation of the response behavior of perchlorate-reducing bacteria to environmental stimuli. It clearly demonstrates attraction toward chlorine oxyanions and the unique ability of these organisms to distinguish structurally analogous compounds, nitrate, chlorate, and perchlorate and respond accordingly. Electronic supplementary material The online version of this article (doi:10.1007/s00253-009-2051-3) contains supplementary material, which is available to authorized users.