Practical Applications of Biofluorescent Particle Counting in Environmental Monitoring Investigations.

Investigations into environmental monitoring (EM) excursions can be prolonged and do not always result in clear root causes or corrective and preventative actions. This article outlines how biofluorescent particle counting (BFPC) can be used in investigations to eliminate the inherent delays of culture-based methods. The application for investigations supplements routine EM, acting as a risk-reduction tool enabling real-time detection of viable microorganisms in air samples and supporting root cause analysis and remedial actions. The article includes guidance on how to use the technology, a real case study involving a mold excursion, and examples of business benefits achieved by various companies.

Characterizing bacterial communities in paper production-troublemakers revealed.

Biofilm formation is a major cause of reduced paper quality and increased down time during paper manufacturing. This study uses Illumina next-generation sequencing to identify the microbial populations causing quality issues due to their presence in biofilms and slimes. The paper defects investigated contained traces of the films and/or slime of mainly two genera, Tepidimonas and Chryseobacterium. The Tepidimonas spp. found contributed on average 68% to the total bacterial population. Both genera have been described previously to be associated with biofilms in paper mills. There was indication that Tepidimonas spp. were present as compact biofilm in the head box of one paper machine and was filtered out by the paper web during production. On the other hand Tepidimonas spp. were also present to a large extent in the press and white waters of two nonproblematic paper machines. Therefore, the mere presence of a known biofilm producer alone is not sufficient to cause slimes and therefore paper defects and other critical factors are additionally at play. For instance, we identified Acidovorax sp., which is an early colonizer of paper machines, exhibiting the ability to form extracellular DNA matrices for attachment and biofilm formation.

Isolation of Legionella-containing vacuoles by immuno-magnetic separation.

The environmental bacterium Legionella pneumophila naturally parasitizes free-living amoebae. L. pneumophila is an opportunistic human pathogen that grows in macrophages, thus causing a life-threatening pneumonia termed Legionnaires' disease. The bacteria replicate intracellularly in environmental and immune phagocytes within a unique compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is a complex and robust process involving >150 secreted bacterial effector proteins, which are believed to subvert host cell signaling and vesicle trafficking pathways. This unit describes a simple approach to purify intact LCVs from Dictyostelium discoideum amoebae. The method comprises a two-step purification protocol that includes immuno-magnetic separation by means of an antibody against an effector protein specifically binding to LCVs, followed by density gradient centrifugation. The use of D. discoideum producing a fluorescent LCV marker and fluorescently labeled L. pneumophila allow tracking the enrichment of LCVs by light microscopy.

Endosomal and secretory markers of the Legionella-containing vacuole.

The Gram-negative opportunistic pathogen Legionella pneumophila replicates in phagocytes within a specific compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is a complex process requiring the bacterial Icm/Dot type IV secretion system and more than 100 translocated effector proteins, which putatively subvert cellular signaling and vesicle trafficking pathways. Phosphoinositide (PI) glycerolipids are pivotal regulators of signal transduction and membrane dynamics in eukaryotes. Recently, a number of Icm/Dot substrates were found to anchor to the LCV membrane by binding to PIs. One of these effectors, SidC, specifically interacts with phosphatidylinositol-4 phosphate [PtdIns(4)P]. Using an antibody against SidC and magnetic beads coupled to a secondary antibody, intact LCVs were purified by immuno-magnetic separation, followed by density centrifugation. This purification strategy is in principle applicable to any pathogen vacuole that carries specific markers. The LCV proteome determined by LC-MS/MS revealed 566 host proteins, including novel components of the endosomal pathway, as well as the early and late secretory trafficking pathways. Thus, LCV formation is a robust process that involves many (functionally redundant) Icm/Dot substrates, as well as the interaction with different host cell vesicle trafficking pathways.

Major surface glycoproteins of insect forms of Trypanosoma brucei are not essential for cyclical transmission by tsetse.

Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the null mutant and wild type trypanosomes (tagged with cyan fluorescent protein) maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the null mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the null mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.

Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila.

The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed "P4M" (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the alpha-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIbeta. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.

Proteome analysis of Legionella vacuoles purified by magnetic immunoseparation reveals secretory and endosomal GTPases.

Legionella pneumophila, the causative agent of Legionnaires' disease, replicates in macrophages and amoebae within 'Legionella-containing vacuoles' (LCVs), which communicate with the early secretory pathway and the endoplasmic reticulum. Formation of LCVs requires the bacterial Icm/Dot type IV secretion system. The Icm/Dot-translocated effector protein SidC selectively anchors to LCVs by binding the host lipid phosphatidylinositol-4-phosphate (PtdIns(4)P). Here, we describe a novel and simple approach to purify intact vacuoles formed by L. pneumophila within Dictyostelium discoideum by using magnetic immunoseparation with an antibody against SidC, followed by density gradient centrifugation. To monitor LCV purification by fluorescence microscopy, we used Dictyostelium producing the LCV marker calnexin-GFP and L. pneumophila labeled with the red fluorescent protein DsRed. A proteome analysis of purified LCVs by liquid chromatography coupled to tandem mass spectrometry revealed 566 host proteins, including known LCV components, such as the small GTPases Arf1, Rab1 and Rab7. Rab8, an endosomal regulator of the late secretory pathway originating from the trans Golgi network, and the endosomal GTPase Rab14 were identified as novel LCV components, which were found to be present on vacuoles harboring wild-type but not Icm/Dot-deficient L. pneumophila. Thus, LCVs also communicate with the late secretory and endosomal pathways. Depletion of Rab8 or Arf1 by RNA interference reduced the amount of SidC on LCVs, indicating that the GTPases promote the recruitment of Legionella effectors by regulating the level of PtdIns(4)P.

The Legionella pneumophila phosphatidylinositol-4 phosphate-binding type IV substrate SidC recruits endoplasmic reticulum vesicles to a replication-permissive vacuole.

Legionella pneumophila, the causative agent of Legionnaires' disease, uses the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system to establish within amoebae and macrophages an endoplasmic reticulum (ER)-derived replication-permissive compartment, the Legionella-containing vacuole (LCV). The Icm/Dot substrate SidC and its paralogue SdcA anchor to LCVs via phosphatidylinositol-4 phosphate [PtdIns(4)P]. Here we identify the unique 20 kDa PtdIns(4)P-binding domain of SidC, which upon heterologous expression in Dictyostelium binds to LCVs and thus is useful as a PtdIns(4)P-specific probe. LCVs harbouring L. pneumophilaDeltasidC-sdcA mutant bacteria recruit ER and ER-derived vesicles less efficiently and carry endosomal but not lysosomal markers. The phenotypes are complemented by supplying sidC on a plasmid. L. pneumophilaDeltasidC-sdcA grows at wild-type rate in calnexin-negative LCVs, suggesting that communication with the ER is dispensable for establishing a replicative compartment. The amount of SidC and calnexin is directly proportional on isolated LCVs, and in a cell-free system, the recruitment of calnexin-positive vesicles to LCVs harbouring DeltasidC-sdcA mutant bacteria is impaired. Beads coated with purified SidC or its 70 kDa N-terminal fragment recruit ER vesicles in Dictyostelium and macrophage lysates. Our results establish SidC as an L. pneumophila effector protein, which anchors to PtdIns(4)P on LCVs and recruits ER vesicles to a replication-permissive vacuole.

Environmental predators as models for bacterial pathogenesis.

Environmental bacteria are constantly threatened by bacterivorous predators such as free-living protozoa and nematodes. In the course of their coevolution with environmental predators, some bacteria developed sophisticated defence mechanisms, including the secretion of toxins, or the capacity to avoid lysosomal killing and to replicate intracellularly within protozoa. To analyse the interactions with bacterial pathogens on a molecular, cellular or organismic level, protozoa and other non-mammalian hosts are increasingly used. These include amoebae, as well as genetically tractable hosts, such as the social amoeba Dictyostelium discoideum, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Using these hosts, the virulence mechanisms of opportunistic pathogenic bacteria such as Legionella, Mycobacterium, Pseudomonas or Vibrio were found to be not only relevant for the interactions of the bacteria with protozoa, nematodes and insect phagocytes, but also with mammalian hosts including humans. Thus, non-mammalian model hosts provide valuable insight into the pathogenesis of environmental bacteria.

A family of stage-specific alanine-rich proteins on the surface of epimastigote forms of Trypanosoma brucei.

A 'two coat' model of the life cycle of Trypanosoma brucei has prevailed for more than 15 years. Metacyclic forms transmitted by infected tsetse flies and mammalian bloodstream forms are covered by variant surface glycoproteins. All other life cycle stages were believed to have a procyclin coat, until it was shown recently that epimastigote forms in tsetse salivary glands express procyclin mRNAs without translating them. As epimastigote forms cannot be cultured, a procedure was devised to compare the transcriptomes of parasites in different fly tissues. Transcripts encoding a family of glycosylphosphatidyl inositol-anchored proteins, BARPs (previously called bloodstream alanine-rich proteins), were 20-fold more abundant in salivary gland than midgut (procyclic) trypanosomes. Anti-BARP antisera reacted strongly and exclusively with salivary gland parasites and a BARP 3' flanking region directed epimastigote-specific expression of reporter genes in the fly, but inhibited expression in bloodstream and procyclic forms. In contrast to an earlier report, we could not detect BARPs in bloodstream forms. We propose that BARPs form a stage-specific coat for epimastigote forms and suggest renaming them brucei alanine-rich proteins.

Expression of procyclin mRNAs during cyclical transmission of Trypanosoma brucei.

Trypanosoma brucei, the parasite causing human sleeping sickness, relies on the tsetse fly for its transmission. In the insect, EP and GPEET procyclins are the major surface glycoproteins of procyclic (midgut) forms of the parasite, with GPEET predominating in the early procyclic form and two isoforms of EP in the late procyclic form. EP procyclins were previously detected on salivary gland trypanosomes, presumably epimastigotes, by immunoelectron microscopy. However, no procyclins could be detected by mass spectrometry when parasites were isolated from infected glands. We have used qualitative and quantitative RT-PCR to analyse the procyclin mRNAs expressed by trypanosomes in the tsetse midgut and salivary glands at different time points after infection. The coding regions of the three EP isoforms (EP1, EP2 and EP3) are extremely similar, but their 3' untranslated regions contain unique sequences that make it possible to assign the cDNAs amplified by this technique. With the exception of EP2, we found that the spectrum of procyclin mRNAs expressed in the midgut mirrors the protein repertoire of early and established procyclic forms. Surprisingly, procyclin mRNAs, including that of GPEET, are present at relatively high levels in salivary gland trypanosomes, although the proteins are rarely detected by immunofluorescence. Additional experiments using transgenic trypanosomes expressing reporter genes or mutant forms of procyclin point to a mechanism of translational or post-translational control, involving the procyclin coding regions, in salivary gland trypanosomes. It is widely accepted that T. brucei always has a coat of either variant surface glycoprotein or procyclin. It has been known for many years that the epimastigote form does not have a variant surface glycoprotein coat. The finding that this life cycle stage is usually negative for procyclin as well is new, and means that the paradigm will need to be revised.