The Coxiella burnetii T4SS Effector AnkF Is Important for Intracellular Replication.

Coxiella burnetii is an obligate intracellular pathogen and the causative agent of the zoonotic disease Q fever. Following uptake by alveolar macrophages, the pathogen replicates in an acidic phagolysosomal vacuole, the C. burnetii-containing vacuole (CCV). Effector proteins translocated into the host cell by the type IV secretion system (T4SS) are important for the establishment of the CCV. Here we focus on the effector protein AnkF and its role in establishing the CCV. The C. burnetii AnkF knock out mutant invades host cells as efficiently as wild-type C. burnetii, but this mutant is hampered in its ability to replicate intracellularly, indicating that AnkF might be involved in the development of a replicative CCV. To unravel the underlying reason(s), we searched for AnkF interactors in host cells and identified vimentin through a yeast two-hybrid approach. While AnkF does not alter vimentin expression at the mRNA or protein levels, the presence of AnkF results in structural reorganization and vesicular co-localization with recombinant vimentin. Ectopically expressed AnkF partially accumulates around the established CCV and endogenous vimentin is recruited to the CCV in a time-dependent manner, suggesting that AnkF might attract vimentin to the CCV. However, knocking-down endogenous vimentin does not affect intracellular replication of C. burnetii. Other cytoskeletal components are recruited to the CCV and might compensate for the lack of vimentin. Taken together, AnkF is essential for the establishment of the replicative CCV, however, its mode of action is still elusive.

The inhibition of the apoptosis pathway by the Coxiella burnetii effector protein CaeA requires the EK repetition motif, but is independent of survivin.

ABSRTACT Coxiella burnetii is an obligate intracellular bacterium that causes Query (Q) fever, a zoonotic disease. It requires a functional type IV secretion system (T4SS) which translocate bacterial effector proteins into the host cell cytoplasm and thereby facilitates bacterial replication. To date, more than 130 effector proteins have been identified, but their functions remain largely unknown. Recently, we demonstrated that one of these proteins, CaeA (CBU1524) localized to the host cell nucleus and inhibited intrinsic apoptosis of HEK293 or CHO cells. In the present study we addressed the question whether CaeA also affects the extrinsic apoptosis pathway. Ectopic expression of CaeA reduced extrinsic apoptosis and prevented the cleavage of the executioner caspase 7, but did not impair the activation of initiator caspase 9. CaeA expression resulted in an up-regulation of survivin (an inhibitor of activated caspases), which, however, was not causal for the anti-apoptotic effect of CaeA. Comparing the sequence of CaeA from 25 different C. burnetii isolates we identified an EK (glutamic acid/ lysine) repetition motif as a site of high genetic variability. The EK motif of CaeA was essential for the anti-apoptotic activity of CaeA. From these data, we conclude that the C. burnetii effector protein CaeA interferes with the intrinsic and extrinsic apoptosis pathway. The process requires the EK repetition motif of CaeA, but is independent of the upregulated expression of survivin.

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

Antiapoptotic activity of Coxiella burnetii effector protein AnkG is controlled by p32-dependent trafficking.

Intracellular bacterial pathogens frequently inhibit host cell apoptosis to ensure survival of their host, thereby allowing bacterial propagation. The obligate intracellular pathogen Coxiella burnetii displays antiapoptotic activity which depends on a functional type IV secretion system (T4SS). Accordingly, antiapoptotic T4SS effector proteins, like AnkG, have been identified. AnkG inhibits pathogen-induced apoptosis, possibly by binding to the host cell mitochondrial protein p32 (gC1qR). However, the molecular mechanism of AnkG activity remains unknown. Here, we demonstrate that ectopically expressed AnkG associates with mitochondria and traffics into the nucleus after apoptosis induction, although AnkG lacks a predicted nuclear localization signal. We identified the p32 interaction region in AnkG and constructed an AnkG mutant (AnkGR(22/23S)) unable to bind to p32. By using this mutant, we found that intracellular localization and trafficking of AnkG into the nucleus are dependent on binding to p32. Furthermore, we demonstrated that nuclear localization of AnkG but not binding to p32 is required for apoptosis inhibition. Thus, the antiapoptotic activity of AnkG is controlled by p32-mediated intracellular trafficking, which, in turn, seems to be regulated by host cell processes that sense stress.

Attachment of capsular polysaccharide to the cell wall in Streptococcus pneumoniae.

Streptococcus pneumoniae protects itself from components of the human immune defense system by a thick polysaccharide capsule, which in most serotypes is covalently attached to the cell wall peptidoglycan. Members of the LytR-Cps2A-Psr (LCP) protein family have recently been implicated in the attachment of anionic polymers to peptidoglycan in Gram-positive bacteria, based on genetic evidence from Bacillus subtilis mutant strains and on the crystal structure of S. pneumoniae Cps2A containing a tightly bound polyprenol (pyro)phosphate lipid. Here, we provide evidence that Cps2A and its two pneumococcal homologs, LytR and Psr, contribute to the maintenance of normal capsule levels and to the retention of the capsular polysaccharide at the cell wall in the capsular type 2 S. pneumoniae strain D39. GFP fusions of all three LCP proteins showed enhanced localization at mid-cell, indicating a role in cell wall growth. Single cps2A or psr mutants produced a reduced amount of capsule. A cps2A lytR double mutant showed greatly impaired growth and cell morphology and lost approximately half of the total capsule material into the culture supernatant. We also present the crystal structure of the B. subtilis LCP protein YwtF and provide crystallographic evidence for the phosphotransferase activity of Cps2A, supporting an enzymatic function in the attachment of capsular polysaccharides to cell wall peptidoglycan.