Metabolic Plasticity Aids Amphotropism of Coxiella burnetii

Coxiella burnetii , the causative agent of Query (Q) fever in humans, is an obligate intracellular bacterium. C. burnetii can naturally infect a broad range of host organisms (e.g., mammals and arthropods) and cell types. This amphotropic nature of C. burnetii , in combination with its ability to utilize both glycolytic and gluconeogenic carbon sources, suggests that the pathogen relies on metabolic plasticity to replicate in nutritionally diverse intracellular environments. To test the significance of metabolic plasticity in C. burnetii host cell colonization, C. burnetii intracellular replication in seven distinct cell lines was compared between a metabolically competent parental strain and a mutant, Cb Δ pckA, unable to undergo gluconeogenesis. Both the parental strain and Cb Δ pckA exhibited host cell-dependent infection phenotypes, which were influenced by alterations to host glycolytic or gluconeogenic substrate availability. Because the nutritional environment directly impacts host cell physiology, our analysis was extended to investigate the response of C. burnetii replication in mammalian host cells cultivated in a novel physiological medium based on the nutrient composition of mammalian interstitial fluid, Interstitial Fluid-modeled Medium (IFmM). An infection model based on IFmM resulted in exacerbation of a replication defect exhibited by Cb Δ pckA in specific cell lines. Cb Δ pckA was also attenuated during infection of an animal host. Overall, the study underscores that gluconeogenic capacity aids C. burnetii amphotropism and that the amphotropic nature of C. burnetii should be considered when resolving virulence mechanisms in this pathogen.

Download Full-text

Identification and Functional Assessment of the First Placental Adhesin of Treponema pallidum That May Play Critical Role in Congenital Syphilis

Frontiers in Microbiology ◽

10.3389/fmicb.2020.621654 ◽

2020 ◽

Vol 11 ◽

Author(s):

Shekerah Primus ◽

Sandra C. Rocha ◽

Lorenzo Giacani ◽

Nikhat Parveen

Keyword(s):

Cell Lines ◽

Dermatan Sulfate ◽

Congenital Syphilis ◽

Treponema Pallidum ◽

Bacterial Attachment ◽

Host Cells ◽

Mammalian Host ◽

Bewo Cell ◽

Specific Cell ◽

Mammalian Cell Lines

Syphilis is a global, re-emerging sexually transmitted infection and congenital syphilis remains a major cause of adverse pregnancy outcomes due to bacterial infection in developing nations with a high rate of fetus loss. The molecular mechanisms involved in pathogenesis of the causative agent, Treponema pallidum subsp. pallidum remain poorly understood due to the difficulties of working with this pathogen, including the inability to grow it in pure culture. To reduce the spread of syphilis, we must first increase our knowledge of the virulence factors of T. pallidum and their contribution to syphilis manifestations. Tp0954 was predicted to be a surface lipoprotein of T. pallidum. Therefore, we experimentally demonstrated that Tp0954 is indeed a surface protein and further investigated its role in mediating bacterial attachment to various mammalian host cells. We found that expression of Tp0954 in a poorly adherent, but physiologically related derivative strain of the Lyme disease causing spirochete Borrelia burgdorferi B314 strain promotes its binding to epithelial as well as non-epithelial cells including glioma and placental cell lines. We also found that Tp0954 expression facilitates binding of this strain to purified dermatan sulfate and heparin, and also that bacterial binding to mammalian cell lines is mediated by the presence of heparan sulfate and dermatan sulfate in the extracellular matrix of the specific cell lines. These results suggest that Tp0954 may be involved not only in initiating T. pallidum infection by colonizing skin epithelium, but it may also contribute to disseminated infection and colonization of distal tissues. Significantly, we found that Tp0954 promotes binding to the human placental choriocarcinoma BeWo cell line, which is of trophoblastic endocrine cell type, as well as human placental tissue sections, suggesting its role in placental colonization and possible contribution to transplacental transmission of T. pallidum. Altogether, these novel findings offer an important step toward unraveling syphilis pathogenesis, including placental colonization and T. pallidum vertical transmission from mother to fetus during pregnancy.

Download Full-text

Perturbation of host autophagy by the Irish potato famine pathogen

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314091736 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C826-C826

Author(s):

Abbas Maqbool ◽

Richard Richard ◽

Tolga Bozkurt ◽

Yasin Dagdas ◽

Khaoula Belhai ◽

...

Keyword(s):

Host Cell ◽

Plant Cells ◽

Irish Potato ◽

Effector Proteins ◽

Host Cells ◽

Cell Physiology ◽

Biochemical Basis ◽

The Impact ◽

Potato Famine ◽

Irish Potato Famine

Autophagy is a catabolic process involving degradation of dysfunctional cytoplasmic components to ensure cellular survival under starvation conditions. The process involves formation of double-membrane vesicles called autophagosomes and delivery of the inner constituents to lytic compartments. It can also target invading pathogens, such as intracellular bacteria, for destruction and is thus implicated in innate immune pathways [1]. In response, certain mammalian pathogens deliver effector proteins into host cells that inhibit autophagy and contribute to enabling parasitic infection [2]. Pyhtophthora infestans, the Irish potato famine pathogen, is a causative agent of late blight disease in potato and tomato crops. It delivers a plethora of modular effector proteins into plant cells to promote infection. Once inside the cell, RXLR-type effector proteins engage with host cell proteins, to manipulate host cell physiology for the benefit of the pathogen. As plants lack an adaptive immune system, this provides a robust mechanism for pathogens to circumvent host defense. PexRD54 is an intracellular RXLR-type effector protein produced by P. infestans. PexRD54 interacts with potato homologues of autophagy protein ATG8 in plant cells. We have been investigating the structural and biochemical basis of the PexRD54/ATG8 interaction in vitro. We have purified PexRD54 and ATG8 independently and in complex from E. coli. Using protein/protein interaction studies we have shown that PexRD54 binds ATG8 with sub-micromolar affinity. We have also determined the structure of PexRD54 in the presence of ATG8. This crystal structure provides key insights into how the previously reported WY-fold of oomycete RXLR-type effectors [3] can be organized in multiple repeats. The structural data also provides insights into the interaction between PexRD54 and ATG8, suggesting further experiments to understand the impact of this interaction on host cell physiology and how this benefits the pathogen.

Download Full-text

Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms

Anais da Academia Brasileira de Ciências ◽

10.1590/s0001-37652005000100006 ◽

2005 ◽

Vol 77 (1) ◽

pp. 77-94 ◽

Cited By ~ 39

Author(s):

Renato A. Mortara ◽

Walter K. Andreoli ◽

Noemi N. Taniwaki ◽

Adriana B. Fernandes ◽

Claudio V. da Silva ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Host Cell ◽

Mammalian Cells ◽

Parasitophorous Vacuole ◽

Host Cells ◽

Mammalian Host ◽

Target Cells ◽

Sylvatic Cycle ◽

Final Destination ◽

Different Strains

Trypanosoma cruzi, the etiological agent of Chagas’ disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm.

Download Full-text

Drug Discovery for Chagas Disease: Impact of Different Host Cell Lines on Assay Performance and Hit Compound Selection

Tropical Medicine and Infectious Disease ◽

10.3390/tropicalmed4020082 ◽

2019 ◽

Vol 4 (2) ◽

pp. 82 ◽

Cited By ~ 5

Author(s):

Caio Haddad Franco ◽

Laura Maria Alcântara ◽

Eric Chatelain ◽

Lucio Freitas-Junior ◽

Carolina Borsoi Moraes

Keyword(s):

Drug Discovery ◽

Host Cell ◽

Cell Lines ◽

Chagas Disease ◽

Host Cells ◽

Mammalian Cell Lines ◽

Starting Point ◽

Disease Impact ◽

Screening Assays ◽

Host Parasite

Cell-based screening has become the major compound interrogation strategy in Chagas disease drug discovery. Several different cell lines have been deployed as host cells in screening assays. However, host cell characteristics and host-parasite interactions may play an important role when assessing anti-T. cruzi compound activity, ultimately impacting on hit discovery. To verify this hypothesis, four distinct mammalian cell lines (U2OS, THP-1, Vero and L6) were used as T. cruzi host cells in High Content Screening assays. Rates of infection varied greatly between different host cells. Susceptibility to benznidazole also varied, depending on the host cell and parasite strain. A library of 1,280 compounds was screened against the four different cell lines infected with T. cruzi, resulting in the selection of a total of 82 distinct compounds as hits. From these, only two hits were common to all four cell lines assays (2.4%) and 51 were exclusively selected from a single assay (62.2%). Infected U2OS cells were the most sensitive assay, as 55 compounds in total were identified as hits; infected THP-1 yielded the lowest hit rates, with only 16 hit compounds. Of the selected hits, compound FPL64176 presented selective anti-T. cruzi activity and could serve as a starting point for the discovery of new anti-chagasic drugs.

Download Full-text

Inhibition of Trypanosoma cruzi growth in mammalian cells by purine and pyrimidine analogs.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.11.2455 ◽

1996 ◽

Vol 40 (11) ◽

pp. 2455-2458 ◽

Cited By ~ 47

Author(s):

J Nakajima-Shimada ◽

Y Hirota ◽

T Aoki

Keyword(s):

Trypanosoma Cruzi ◽

Growth Inhibition ◽

Host Cell ◽

Culture System ◽

Mammalian Cells ◽

Screening Method ◽

Host Cells ◽

Mammalian Host ◽

Dependent Manner ◽

Pyrimidine Analogs

Trypanosoma cruzi, the causative agent of Chagas' disease, exhibits two different developmental stages in mammals, the amastigote, an intracellular form that proliferates in the cytoplasm of host cells, and the trypomastigote, an extracellular form that circulates in the bloodstream. We have already established an in vitro culture system using mammalian host cells (HeLa) infected with T. cruzi in which the time course of parasite growth is determined quantitatively. We adopted this system for the screening of anti-T. cruzi agents that would ideally prove to be effective against trypanosomes with no toxicity to the host cell. Of the purine analogs tested, allopurinol markedly inhibited the growth of amastigotes in a dose-dependent manner, with no lethal effect on trypomastigotes. 3'-Deoxyinosine and 3'-deoxyadenosine also suppressed T. cruzi growth inside the host cell, with the concentrations causing 50% growth inhibition being 10 and 5 microM, respectively, in contrast to a concentration causing 50% growth inhibition of 3 microM for allopurinol. Among the pyrimidine analogs examined, 3'-azido-3'-deoxythymidine (zidovudine) significantly reduced the growth of the parasite at concentrations as low as 1 microM. The anti-human immunodeficiency virus agents 2',3'-dideoxyinosine and 2',3'-dideoxyadenosine caused a decrease in amastigote growth, while 2',3'-dideoxycytidine and 2',3'-dideoxyuridine had no inhibitory effect. When Swiss 3T3 fibroblasts were used as host cells, allopurinol, 3'-deoxyinosine, 3'-deoxyadenosine, and 3'-azid-3'-deoxythymidine also markedly inhibited T. cruzi proliferation. These results indicate that our culture system is useful as a primary screening method for candidate compounds against T. cruzi on the basis of two criteria, namely, intracellular replication by the parasite and host-cell infection rate.

Download Full-text

Characterization of Sec-Translocon-Dependent Extracytoplasmic Proteins of Rickettsia typhi

Journal of Bacteriology ◽

10.1128/jb.00794-08 ◽

2008 ◽

Vol 190 (18) ◽

pp. 6234-6242 ◽

Cited By ~ 19

Author(s):

Nicole C. Ammerman ◽

M. Sayeedur Rahman ◽

Abdu F. Azad

Keyword(s):

Host Cell ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Host Cells ◽

Transcriptional Analysis ◽

Mammalian Host ◽

Signal Peptides ◽

Cell Infection ◽

Rickettsia Typhi ◽

Sec Translocon

ABSTRACT As obligate intracellular, vector-borne bacteria, rickettsiae must adapt to both mammalian and arthropod host cell environments. Deciphering the molecular mechanisms of the interactions between rickettsiae and their host cells has largely been hindered by the genetic intractability of these organisms; however, research in other gram-negative pathogens has demonstrated that many bacterial determinants of attachment, entry, and pathogenesis are extracytoplasmic proteins. The annotations of several rickettsial genomes indicate the presence of homologs of the Sec translocon, the major route for bacterial protein secretion from the cytoplasm. For Rickettsia typhi, the etiologic agent of murine typhus, homologs of the Sec-translocon-associated proteins LepB, SecA, and LspA have been functionally characterized; therefore, the R. typhi Sec apparatus represents a mechanism for the secretion of rickettsial proteins, including virulence factors, into the extracytoplasmic environment. Our objective was to characterize such Sec-dependent R. typhi proteins in the context of a mammalian host cell infection. By using the web-based programs LipoP, SignalP, and Phobius, a total of 191 R. typhi proteins were predicted to contain signal peptides targeting them to the Sec translocon. Of these putative signal peptides, 102 were tested in an Escherichia coli-based alkaline phosphatase (PhoA) gene fusion system. Eighty-four of these candidates exhibited signal peptide activity in E. coli, and transcriptional analysis indicated that at least 54 of the R. typhi extracytoplasmic proteins undergo active gene expression during infections of HeLa cells. This work highlights a number of interesting proteins possibly involved in rickettsial growth and virulence in mammalian cells.

Download Full-text

Effect of Host Cells on Low- and Medium-Pressure UV Inactivation of Adenoviruses

Applied and Environmental Microbiology ◽

10.1128/aem.00185-10 ◽

2010 ◽

Vol 76 (21) ◽

pp. 7068-7075 ◽

Cited By ~ 21

Author(s):

Huiling Guo ◽

Xiaona Chu ◽

Jiangyong Hu

Keyword(s):

Host Cell ◽

Cell Line ◽

Cell Lines ◽

Host Cells ◽

Uv Disinfection ◽

Human Embryonic Kidney ◽

Experimental Conditions ◽

Medium Pressure ◽

Serotype 5 ◽

Uv Dose

ABSTRACT UV disinfection is highly effective against most pathogens, with the exception of the adenoviruses (AD). To date, many studies have focused on low-pressure (LP) UV inactivation of AD, but little is known about the effect of medium-pressure (MP) UV inactivation of AD. Despite numerous studies of LP UV inactivation of AD, extreme variabilities in the LP UV dose requirements of AD had been observed because of differing experimental conditions used, such as the types of cell lines used for AD enumeration. This study therefore investigates the effect of three different host cell lines (PLC/PRF/5, human embryonic kidney 293 [HEK293], and XP17BE) on the LP and MP UV dose requirements of AD serotype 5 (AD5), AD40, and AD41 under similar experimental settings. Results showed that for 4-log inactivation of AD, LP UV and MP UV doses needed to be in the ranges of 123 to 182 mJ/cm2 and 65 to 90 mJ/cm2, respectively, when HEK293 and PLC/PRF/5 cells were used for enumeration. The UV doses required for MP UV inactivation of AD were significantly lower than those required for LP UV inactivation (P value < 0.05). When different cell lines were used for enumeration, UV dose requirements for AD differed. AD were portrayed to be most susceptible to UV (LP UV doses of <57 mJ/cm2 and MP UV doses of <42 mJ/cm2 for 4-log AD inactivation) when the XP17BE cells were used as the host cell. The use of different cell lines for AD enumeration affected LP UV dose results more significantly than MP UV dose results (P value < 0.05). Cell line variability factors for LP UV disinfection (CLLP) and MP UV disinfection (CLMP) for AD5, AD40, and AD41 enumerated with HEK293, PLC/PRF/5, and XP17BE cells were in the ranges of 1.0 to 3.2 and 1.0 to 2.5, respectively.

Download Full-text

Critical Role for Molecular Iron in Coxiella burnetii Replication and Viability

mSphere ◽

10.1128/msphere.00458-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Savannah E. Sanchez ◽

Anders Omsland

Keyword(s):

Host Cell ◽

Coxiella Burnetii ◽

Cultured Cells ◽

Critical Role ◽

Host Cells ◽

Physiological Adaptation ◽

Iron Starvation ◽

Iron Storage ◽

Content Type ◽

Forms Of Iron

ABSTRACT Coxiella burnetii, the causative agent of Query (Q) fever in humans, is a highly infectious obligate intracellular bacterium. Following uptake into a host cell, C. burnetii replicates within a phagolysosome-derived compartment referred to as the Coxiella-containing vacuole (CCV). During infection, C. burnetii exhibits tropism for tissues related to iron storage and recycling (e.g., the liver and splenic red pulp), suggesting that pathogen physiology is linked to host iron metabolism. Iron has been described to have a limited role in C. burnetii virulence regulation, despite evidence that C. burnetii-infected host cells increase expression of transferrin receptors, thereby suggesting that active iron acquisition by the bacterium occurs upon infection. Through the use of host cell-free culture, C. burnetii was separated from the host cell in order to directly assess the role of different forms of iron in C. burnetii replication and viability, and therefore virulence. Results indicate that C. burnetii tolerates molecular iron over a broad concentration range (i.e., ∼0.001 to 1 mM) and undergoes gross loss of viability upon iron starvation. C. burnetii protein synthesis and energy metabolism, however, occur nearly uninhibited under iron concentrations not permissive to replication. Despite the apparent absence of genes related to acquisition of host-associated iron-containing proteins, C. burnetii replication is supported by hemoglobin, transferrin, and ferritin, likely due to release of iron from such proteins under acidic conditions. Moreover, chelation of host iron pools inhibited pathogen replication during infection of cultured cells. IMPORTANCE Host organisms restrict the availability of iron to invading pathogens in order to reduce pathogen replication. To counteract the host’s response to infection, bacteria can rely on redundant mechanisms to obtain biologically diverse forms of iron during infection. C. burnetii appears specifically dependent on molecular iron for replication and viability and exhibits a response to iron akin to bacteria that colonize iron-rich environments. Physiological adaptation of C. burnetii to the unique acidic and degradative environment of the CCV is consistent with access of this pathogen to molecular iron.

Download Full-text

Dependency of Coxiella burnetii Type 4B Secretion on the Chaperone IcmS

Journal of Bacteriology ◽

10.1128/jb.00431-19 ◽

2019 ◽

Vol 201 (23) ◽

Cited By ~ 2

Author(s):

Charles L. Larson ◽

Paul A. Beare ◽

Robert A. Heinzen

Keyword(s):

Host Cell ◽

Coxiella Burnetii ◽

Legionella Pneumophila ◽

Q Fever ◽

Secretion System ◽

Host Cells ◽

Growth Defect ◽

Wild Type ◽

Signal Sequences ◽

Content Type

ABSTRACT Macrophage parasitism by Coxiella burnetii, the cause of human Q fever, requires the translocation of proteins with effector functions directly into the host cell cytosol via a Dot/Icm type 4B secretion system (T4BSS). Secretion by the analogous Legionella pneumophila T4BSS involves signal sequences within the C-terminal and internal domains of effector proteins. The cytoplasmic chaperone pair IcmSW promotes secretion and binds internal sites distinct from signal sequences. In the present study, we investigated requirements of C. burnetii IcmS for host cell parasitism and effector translocation. A C. burnetii icmS deletion mutant (ΔicmS) exhibited impaired replication in Vero epithelial cells, deficient formation of the Coxiella-containing vacuole, and aberrant T4BSS secretion. Three secretion phenotypes were identified from a screen of 50 Dot/Icm substrates: IcmS dependent (secreted by only wild-type bacteria), IcmS independent (secreted by both wild-type and ΔicmS bacteria), or IcmS inhibited (secreted by only ΔicmS bacteria). Secretion was assessed for N-terminal or C-terminal truncated forms of CBU0794 and CBU1525. IcmS-inhibited secretion of CBU1525 required a C-terminal secretion signal whereas IcmS-dependent secretion of CBU0794 was directed by C-terminal and internal signals. Interchange of the C-terminal 50 amino acids of CBU0794 and CBU1525 revealed that sites within the C terminus regulate IcmS dependency. Glutathione S-transferase-tagged IcmSW bound internal sequences of IcmS-dependent and -inhibited substrates. Thus, the growth defect of the C. burnetii ΔicmS strain is associated with a loss of T4BSS chaperone activity that both positively and negatively regulates effector translocation. IMPORTANCE The intracellular pathogen Coxiella burnetii employs a type 4B secretion system (T4BSS) that promotes growth by translocating effectors of eukaryotic pathways into host cells. T4BSS regulation modeled in Legionella pneumophila indicates IcmS facilitates effector translocation. Here, we characterized type 4B secretion by a Coxiella ΔicmS mutant that exhibits intracellular growth defects. T4BSS substrates demonstrated increased, equivalent, or decreased secretion by the ΔicmS mutant relative to wild-type Coxiella. Similar to the Legionella T4BSS, IcmS dependency in Coxiella was determined by C-terminal and/or internal secretion signals. However, IcmS inhibited secretion of some effectors by Coxiella that were previously shown to be translocated by Legionella. Thus, Coxiella has a unique IcmS regulatory mechanism that both positively and negatively regulates T4BSS export.

Download Full-text

Gene Expression Changes Induced byTrypanosoma cruziShed Microvesicles in Mammalian Host Cells: Relevance of tRNA-Derived Halves

BioMed Research International ◽

10.1155/2014/305239 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11 ◽

Cited By ~ 52

Author(s):

Maria R. Garcia-Silva ◽

Florencia Cabrera-Cabrera ◽

Roberta Ferreira Cura das Neves ◽

Thaís Souto-Padrón ◽

Wanderley de Souza ◽

...

Keyword(s):

Gene Expression ◽

Host Cell ◽

Extracellular Vesicles ◽

Small Rna ◽

Hela Cells ◽

Small Rnas ◽

Host Cells ◽

Mammalian Host ◽

Large Set ◽

Rna Pathways

At present, noncoding small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, canonical small RNA pathways seem to be lost or excessively simplified in some unicellular organisms includingTrypanosoma cruziwhich lack functional RNAi pathways. Recently, we reported the presence of alternate small RNA pathways inT. cruzimainly represented by homogeneous populations of tRNA- and rRNA-derived small RNAs, which are secreted to the extracellular medium included in extracellular vesicles. Extracellular vesicle cargo could be delivered to other parasites and to mammalian susceptible cells promoting metacyclogenesis and conferring susceptibility to infection, respectively. Here we analyzed the changes in gene expression of host HeLa cells induced by extracellular vesicles fromT. cruzi. As assessed by microarray assays a large set of genes in HeLa cells were differentially expressed upon incorporation ofT. cruzi-derived extracellular vesicles. The elicited response modified mainly host cell cytoskeleton, extracellular matrix, and immune responses pathways. Some genes were also modified by the most abundant tRNA-derived small RNAs included in extracellular vesicles. These data suggest that microvesicles secreted byT. cruzicould be relevant players in early events of theT. cruzihost cell interplay.

Download Full-text