automated-high-throughput-detection-bacterial-adherence-to-host

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in ...

Texas A&M Health Science Center

DNA watermarking of infectious agents: progress and prospects.

Genomic polymorphisms as inherent watermarks for tracking infectious agents.

Many phytopathogenic fungi use infection structures (IFSs, i.e., appressoria and infection cushions) to penetrate host cuticles. However, the conserved mechanisms that mediate initiation of IFS formation in divergent pathogens upon sensing the presence of host plants remain obscure. Here, we demonstrate that a conserved septin gene SEP4 plays crucial roles in this process. Disruption of SEP4 in the plant grey mould fungus Botrytis cinerea completely blocked IFS formation and abolished the virulence of ΔBcsep4 mutants on unwounded hosts. During IFS formation, mutants lacking SEP4 could produce reactive oxygen species (ROS) normally. Inhibition of ROS production in strains harbouring the SEP4 gene resulted in disordered assembly of Sep4 and the subsequent failure to form infection cushions, suggesting that proper Sep4 assembly regulated by ROS is required for initiation of IFS formation and infection. Moreover, loss of SEP4 severely impaired mutant conidiation, melanin and chitin accumulation in hyphal tips and lesion expansion on wounded hosts, but significantly promoted germ tube elongation and sclerotium production. SEP4-mediated fungal pathogenic development, including IFS formation, was validated in the hemibiotroph Magnaporthe oryzae. Our findings indicate that Sep4 plays pleiotropic roles in B. cinerea development and specifically facilities host infection by mediating initiation of IFS formation in divergent plant fungal pathogens in response to ROS signaling.

The septin protein Sep4 facilitates host infection by plant fungal pathogens via mediating initiation of infection structure formation.

Cryptococcus neoformans (Cn) is a deadly fungal pathogen whose intracellular lifestyle is important for virulence. Host mechanisms controlling fungal phagocytosis and replication remain obscure. Here, we perform a global phosphoproteomic analysis of the host response to Cryptococcus infection. Our analysis reveals numerous and diverse host proteins that are differentially phosphorylated following fungal ingestion by macrophages, thereby indicating global reprogramming of host kinase signaling. Notably, phagocytosis of the pathogen activates the host autophagy initiation complex (AIC) and the upstream regulatory components LKB1 and AMPKα, which regulate autophagy induction through their kinase activities. Deletion of Prkaa1, the gene encoding AMPKα1, in monocytes results in resistance to fungal colonization of mice. Finally, the recruitment of AIC components to nascent Cryptococcus-containing vacuoles (CnCVs) regulates the intracellular trafficking and replication of the pathogen. These findings demonstrate that host AIC regulatory networks confer susceptibility to infection and establish a proteomic resource for elucidating host mechanisms that regulate fungal intracellular parasitism.

Global Reprogramming of Host Kinase Signaling in Response to Fungal Infection.

Botrytis cinerea is a necrotrophic plant fungal pathogen that annually causes enormous economic losses worldwide. The ribosome is an organelle for cellular protein biosynthesis. However, little is known about how the ribosome operates as a machine to mediate microbial pathogenesis. Here, we demonstrate that Nop53, a late-acting factor for 60S ribosomal subunit maturation, is crucial for the pathogen's development and virulence. BcNop53 is functionally equivalent to yeast nop53p. Complementation of BcNOP53 completely restored the growth defect of the yeast Δnop53 mutant. BcNop53 is located in nuclei and disruption of BcNOP53 also dramatically impaired pathogen growth. Deletion of BcNOP53 blocked infection structure formation and abolished virulence of the pathogen, possibly due to reduced production of reactive oxygen species. Moreover, loss of BcNOP53 impaired pathogen conidiation and stress adaptation, altered conidial and sclerotial morphology, retarded conidium and sclerotium germination as well as reduced the activities of cell-wall degradation-associated enzymes. Sclerotium production was, however, increased. Complementation with the wild-type BcNOP53 allele rescued defects found in the ΔBcnop53 mutant. Our work establishes a systematic elucidation of Nop53 in regulating microbial development and pathogenesis, provides novel insights into ribosomal processes that regulate fungal pathogenesis, and may open up new targets for addressing fungal diseases.

The pre-rRNA processing factor Nop53 regulates fungal development and pathogenesis via mediating production of reactive oxygen species.

 spp. are intracellular vacuolar pathogens that causes brucellosis, a worldwide zoonosis of profound importance. We previously demonstrated that the activity of host unfolded protein response (UPR) sensor IRE1α (inositol-requiring enzyme 1) and ER-associated autophagy confer susceptibility to  and  intracellular replication. However, the mechanism by which host IRE1α regulates the pathogen intracellular lifestyle remains elusive. In this study, by employing a diverse array of molecular approaches, including biochemical analyses, fluorescence microscopy imaging, and infection assays using primary cells derived from  (encoding IRE1) conditional knockout mice, we address this gap in our understanding by demonstrating that a novel IRE1α to ULK1, an important component for autophagy initiation, signaling axis confers susceptibility to  intracellular parasitism. Importantly, deletion or inactivation of key signaling components along this axis, including IRE1α, BAK/BAX, ASK1, and JNK as well as components of the host autophagy system ULK1, Atg9a, and Beclin 1, resulted in striking disruption of  intracellular trafficking and replication. Host kinases in the IRE1α-ULK1 axis, including IRE1α, ASK1, JNK1, and/or AMPKα as well as ULK1, were also coordinately phosphorylated in an IRE1α-dependent fashion upon the pathogen infection. Taken together, our findings demonstrate that the IRE1α-ULK1 signaling axis is subverted by the bacterium to promote intracellular parasitism, and provide new insight into our understanding of the molecular mechanisms of intracellular lifestyle of .

Activation of Host IRE1α-Dependent Signaling Axis Contributes the Intracellular Parasitism of .

 is an important causative agent of nosocomial infections worldwide. The pathogen also readily acquires resistance to antibiotics, and pan-resistant strains have been reported.  is widely regarded as an extracellular bacterial pathogen. However, accumulating evidence demonstrates that the pathogen can invade, survive or persist in infected mammalian cells. Unfortunately, the molecular mechanisms controlling these processes remain poorly understood. Here, we show that  S2 cells provide several attractive advantages as a model system for investigating the intracellular lifestyle of the pathogen, including susceptibility to bacterial intracellular replication and limited infection-induced host cell death. We also show that the  system can be used to rapidly identify host factors, including MAP kinase proteins, which confer susceptibility to intracellular parasitism. Finally, analysis of the  system suggested that host proteins that regulate organelle biogenesis and membrane trafficking contribute to regulating the intracellular lifestyle of the pathogen. Taken together, these findings establish a novel model system for elucidating interactions between  and host cells, define new factors that regulate bacterial invasion or intracellular persistence, and identify subcellular compartments in host cells that interact with the pathogen.

A Tractable  Cell System Enables Rapid Identification of  Host Factors.

Phagocytosis and autophagy play critical roles in immune defense. The human fungal pathogen  (Cn) subverts host autophagy-initiation complex (AIC)-related proteins, to promote its phagocytosis and intracellular parasitism of host cells. The mechanisms by which the pathogen engages host AIC-related proteins remain obscure. Here, we show that the recruitment of host AIC proteins to forming phagosomes is dependent upon the activity of CD44, a host cell surface receptor that engages fungal hyaluronic acid (HA). This interaction elevates intracellular Ca concentrations and activates CaMKKβ and its downstream target AMPKα, which results in activation of ULK1 and the recruitment of AIC components. Moreover, we demonstrate that HA-coated beads efficiently recruit AIC components to phagosomes and CD44 interacts with AIC components. Taken together, these findings show that fungal HA plays a critical role in directing the internalization and productive intracellular membrane trafficking of a fungal pathogen of global importance.

Qing-Ming Qin

Department of Microbial Pathogenesis and Immunology

Texas A&M Health Science Center

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells