Intracellular Bacterial Pathogens

Vaccination is the most important approach to counteract infectious diseases. Thus, the development of new and improved vaccines for existing, emerging, and re-emerging diseases is an area of great interest to the scientific community and general public. Traditional approaches to subunit antigen discovery and vaccine development lack consideration for the critical aspects of public safety and activation of relevant protective host immunity. The availability of genomic sequences for pathogenic Brucella spp. and their hosts have led to development of systems-wide analytical tools that have provided a better understanding of host and pathogen physiology while also beginning to unravel the intricacies at the host-pathogen interface. Advances in pathogen biology, host immunology, and host-agent interactions have the potential to serve as a platform for the design and implementation of better-targeted antigen discovery approaches. With emphasis on Brucella spp., we probe the biological as...

Leishmania spp. are sandfly-transmitted parasitic protozoa that cause a spectrum of important diseases and lifelong chronic infections in humans. In the mammalian host, these parasites proliferate within acidified vacuoles in several phagocytic host cells, including macrophages, dendritic cells, and neutrophils. In this review, we discuss recent progress that has been made in defining the nutrient composition of the Leishmania parasitophorous vacuole, as well as metabolic pathways required by these parasites for virulence. Analysis of the virulence phenotype of Leishmania mutants has been particularly useful in defining carbon sources and nutrient salvage pathways that are essential for parasite persistence and/or induction of pathology. We also review data suggesting that intracellular parasite stages modulate metabolic processes in their host cells in order to a generate a more permissive niche.

Proteins that metabolize or bind the nucleotide second messenger cyclic diguanylate regulate a wide variety of important processes in bacteria. These processes include motility, biofilm formation, cell division, differentiation, and virulence. The role of cyclic diguanylate signaling in the lifestyle of Legionella pneumophila, the causative agent of Legionnaires' disease, has not previously been examined. The L. pneumophila genome encodes 22 predicted proteins containing domains related to cyclic diguanylate synthesis, hydrolysis, and recognition. We refer to these genes as cdgS (cyclic diguanylate signaling) genes. Strains of L. pneumophila containing deletions of all individual cdgS genes were created and did not exhibit any observable growth defect in growth medium or inside host cells. However, when overexpressed, several cdgS genes strongly decreased the ability of L. pneumophila to grow inside host cells. Expression of these cdgS genes did not affect the Dot/Icm type IVB secretion system, the major determinant of intracellular growth in L. pneumophila. L. pneumophila strains overexpressing these cdgS genes were less cytotoxic to THP-1 macrophages than wild-type L. pneumophila but retained the ability to resist grazing by amoebae. In many cases, the intracellular-growth inhibition caused by cdgS gene overexpression was independent of diguanylate cyclase or phosphodiesterase activities. Expression of the cdgS genes in a Salmonella enterica serovar Enteritidis strain that lacks all diguanylate cyclase activity indicated that several cdgS genes encode potential cyclases. These results indicate that components of the cyclic diguanylate signaling pathway play an important role in regulating the ability of L. pneumophila to grow in host cells. IMPORTANCE All bacteria must sense and respond to environmental cues. Intracellular bacterial pathogens must detect and respond to host functions that limit their ability to carry out a successful infection. Small-molecule second messengers play key roles in transmitting signals from environmental receptors to the proteins and other components that respond to signals. Cyclic diguanylate is a ubiquitous bacterial second messenger known to play an important role in many sensing and signaling systems in bacteria. The causative agent of Legionnaires' disease, Legionella pneumophila, is an intracellular pathogen that grows inside environmental protists and human macrophages by subverting the normal processes that these cells use to capture and destroy bacteria. We show that the several cyclic diguanylate signaling components in Legionella play a role in the ability to grow inside both kinds of host cells. This work highlights the role of cyclic diguanylate signaling during intracellular growth.

Silver nanoparticles is gaining momentum because of its unique physiochemical properties. Therefore biological methods have been employed for its synthesis. Among such methods mycofabrication is becoming popular which make use of eukaryotic fungi to produce nanoparticles of different morphology either extracellularly or intracellular with the help of an enzyme nitrate reductase

"Background

Symbioses between metazoans and microbes are widespread and vital to many ecosystems. Recent work with several nematode species has suggested that strong associations with microbial symbionts may also be common among members of this phylum. In this work we explore possible symbiosis between bacteria and the free living soil bacteriovorous nematode Acrobeloides maximus.

Methodology

We used a soil microcosm approach to expose A. maximus populations grown monoxenically on RFP labeled Escherichia coli in a soil slurry. Worms were recovered by density gradient separation and examined using both culture-independent and isolation methods. A 16S rRNA gene survey of the worm-associated bacteria was compared to the soil and to a similar analysis using Caenorhabditis elegans N2. Recovered A. maximus populations were maintained on cholesterol agar and sampled to examine the population dynamics of the microbiome.

Results

A consistent core microbiome was extracted from A. maximus that differed from those in the bulk soil or the C. elegans associated set. Three genera, Ochrobactrum, Pedobacter, and Chitinophaga, were identified at high levels only in the A. maximus populations, which were less diverse than the assemblage associated with C. elegans. Putative symbiont populations were maintained for at least 4 months post inoculation, although the levels decreased as the culture aged. Fluorescence in situ hybridization (FISH) using probes specific for Ochrobactrum and Pedobacter stained bacterial cells in formaldehyde fixed nematode guts.

Conclusions

Three microorganisms were repeatedly observed in association with Acrobeloides maximus when recovered from soil microcosms. We isolated several Ochrobactrum sp. and Pedobacter sp., and demonstrated that they inhabit the nematode gut by FISH. Although their role in A. maximus is not resolved, we propose possible mutualistic roles for these bacteria in protection of the host against pathogens and facilitating enzymatic digestion of other ingested bacteria."

Rickettsiae are obligate intracellular bacteria that can cause life-threatening illnesses and are among the oldest known vector-borne pathogens. Members of this genus are extraordinarily diverse and exhibit a broad host range. To establish intracellular infection, Rickettsia species undergo complex, multistep life cycles that are encoded by heavily streamlined genomes. As a result of reductive genome evolution, rickettsiae are exquisitely tailored to their host cell environment but cannot survive extracellularly. This host-cell dependence makes for a compelling system to uncover novel host-pathogen biology, but it has also hindered experimental progress. Consequently, the molecular details of rickettsial biology and pathogenesis remain poorly understood. With recent advances in molecular biology and genetics, the field is poised to start unraveling the molecular mechanisms of these host-pathogen interactions. Here, we review recent discoveries that have shed light on key aspects of rickettsial biology. These studies have revealed that rickettsiae subvert host cells using mechanisms that are distinct from other better-studied pathogens, underscoring the great potential of the Rickettsia genus for revealing novel biology. We also highlight several open questions as promising areas for future study and discuss the path toward solving the fundamental mysteries of this neglected and emerging human pathogen.

Background Quantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections. Therefore, rapid, accurate and low-cost methods represent valuable tools in determining bacterial ability to persist and proliferate within eukaryotic cells. Results Herein, we present the first application of the immunofluorescence In-Cell Western (ICW) assay aimed at quantifying intracellular bacteria in in vitro infection models. The performance of this new approach was evaluated in cell culture infection models using three microorganisms with different lifestyles. Two facultative intracellular bacteria, the fast-growing Shigella flexneri and a persistent strain of Escherichia coli, as well as the obligate intracellular bacterium Chlamydia trachomatis were chosen as bacterial models. The ICW assay was performed in parallel with conventional quantification methods, i.e. colony forming units (CFUs) and inclusion forming units ...

Collagen deposition within granulomas formed after Mycobacterium avium infection was analysed on histological sections stained with Masson’s trichrome using acquired computerized image analysis and a program that was specifically designed for that purpose. Comparison was made between immunocompetent C57BL⁄ 6 mice and mice genetically deficient in the inducible nitric oxide (NO) synthase gene (iNOS– ⁄ – mice) infected with either a highly virulent strain or a moderately virulent strain of M. avium. iNOS-deficient mice were more resistant to the highly virulent strain than control C57B1⁄ 6 mice, but both strains were equally susceptible to the less virulent M. avium strain. Collagen distribution in the granuloma was found in the cuff surrounding the granuloma in an area rich in lymphoid cells as well as inside the granuloma itself, conferring a mesh-like structure within that lesion. It was seen that iNOS– ⁄ – mice induced a higher collagen deposition than C57BL ⁄ 6 mice and that such collagen deposition varied with the mycobacterial strain used to
infect the animals.

The growth in C57Bl/6 mice of seven distinct Mycobacterium avium strains was not exacerbated by the disruption of the inducible (type 2) nitric oxide synthase regardless of the virulence of the strain. The susceptibility of this panel of M. avium strains to reactive nitrogen intermediates in a cell-free system, namely the exposure to acidified nitrite or to the NO donor NOC-18, showed that there is no correlation between strain virulence and the corresponding minimal bactericidal and minimal inhibitory concentrations for those compounds determined in vitro.

The use of nanoparticle delivery systems to enhance intracellular penetration of antibiotics and their retention time is becoming popular. The challenge, however, is that the interaction of nanoparticles with biological systems at the cellular level must be established prior to biomedical applications. Ciprofloxacin-cockle shells-derived calcium carbonate (aragonite) nanoparticles (C-CSCCAN) were developed and characterized. Antibacterial activity was determined using a modified disc diffusion protocol on Salmonella Typhimurium (S. Typhimurium). Biocompatibilittes with macrophage were evaluated using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Bromo-2 1-deoxyuridine (BrdU) assays. Transcriptional regulation of interleukin 1 beta (IL-1β) was determined using reverse transcriptase-polymerase chain reaction (RT-PCR). C-CSCCAN were spherical in shape, with particle sizes ranging from 11.93 to 22.12 nm. Encapsulation efficiency (EE) and loading content (LC) were 99.5% and 5.9%, respectively, with negative ζ potential. X-ray diffraction patterns revealed strong crystallizations and purity in the formulations. The mean diameter of inhibition zone was 18.6˘0.5 mm, which was better than ciprofloxacin alone (11.7˘0.9 mm). Study of biocompatability established the cytocompatability of the delivery system without upregulation of IL-1β. The results indicated that ciprofloxacin-nanoparticles enhanced the antibacterial efficacy of the antibiotic, and could act as a suitable delivery system against intracellular infections.

The peptidoglycan (PG) cell wall is a peptide cross-linked glycan polymer essential for bacterial division and maintenance of cell shape and hydrostatic pressure. Bacteria in the Chlamydiales were long thought to lack PG until recent advances in PG labeling technologies revealed the presence of this critical cell wall component in Chlamydia trachomatis. In this study, we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolution microscopy to demonstrate that four pathogenic Chlamydiae species each possess a 140 nm wide PG ring limited to the division plane during the replicative phase of their developmental cycles. Assembly of this PG ring is rapid, processive, and linked to the bacterial actinlike protein, MreB. Both MreB polymerization and PG biosynthesis occur only in the intracellular form of pathogenic Chlamydia and are required for cell enlargement, division, and transition between the microbe's developmental forms. Our kinetic, molecular, and biochemical analyses suggest that the development of this limited, transient, PG ring structure is the result of pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host.

Extracellular nucleotides are danger signals involved in recognition and control of intracellular pathogens. They are an important component of the innate immune response against intracellular pathogens, inducing the recruitment of inflammatory cells, stimulating secretion of cytokines, and producing inflammatory mediators such as reactive oxygen species (ROS) and nitric oxide (NO). In the case of extracellular ATP, some of the immune responses are mediated through activation of the NLRP3 inflammasome and secretion of the cytokine, interleukin-1b (IL-1b), through a mechanism dependent on ligation of the P2X7 receptor. Here we review the role of extracellular nucleotides as sensors of intracellular bacteria and protozoan parasites, and discuss how these pathogens manipulate purinergic signaling to diminish the immune response against infection.

Polymorphonuclear neutrophil granulocytes (PMN) are primary antimicrobial effector cells of the innate immune system and serve to destroy invading pathogens. Although most ingested microorganisms are killed readily inside PMN, several obligate or facultative intracellular pathogens survive even in this hostile environment. Extension of the life span of neutrophils is a general escape mechanism of pathogens residing in PMN. However, after 2-4 days, even infected neutrophils become apoptotic and are phagocytosed by macrophages. Since microbes entering macrophages via the uptake of infected apoptotic PMN may survive and multiply in macrophages, apoptotic neutrophils can serve as ''Trojan horses'' for certain pathogens. Interfering with activating signaling pathways appears to be another potent mechanism by which intracellular microorganisms suppress cellular activation in neutrophils. In addition to provide a short overview of the topic, the present review aims to summarize our own findings regarding the interaction between human neutrophils and intracellular pathogens as well as regarding the disease promoting role of apoptotic cells after infection with Leishmania major.

Abstract
Details are given in this paper of the development of a numerical model for describing the bacterial transport processes in estuarine and coastal waters.  The transport of enteric bacteria is influenced by many physical, chemical and biological factors, including the sediments contained within the water column and on the seabed.  In recent years, an increasing number of studies have shown that generally the sediments contain much higher bacterial population levels than previously assumed.  The aim of this study has therefore been to develop a numerical modelling tool for predicting the sediment-linked bacterial concentration levels in coastal and estuarine waters.  This paper outlines the theoretical background of an enteric bacterial water quality numerical model.  A conceptual model has been established for representing the transport and of bacteria due to sediment movement, including deposition and suspension.  Details are given of the solute and mass transport equations used to simulate the flow and transport of suspended sediments and enteric bacterial indicators. The enteric bacteria transport equation includes enhanced source and sink terms to represent bacterial kinetic transformation and disappearance or reappearance due to sediment deposition or re-suspension.  The model has been applied to the turbid water environment in the Bristol Channel and Severn Estuary, U.K.  Initial results from the modelling study are presented.