The anti-apoptotic Coxiella burnetii effector protein AnkG is a strain specific virulence factor

Walter Schäfer,João Paulo Gomes,Anja Lührmann,Jonas Holzinger,Julian Pechstein,Paul A Beare,Carsten Heydel,Teresa Schmidt,Arne Cordsmeier,Christian Berens,Jan Schulze-Luehrmann,Vítor Borges,Nicole Wagner

doi:10.1038/s41598-020-72340-9

Abstract

The ability to inhibit host cell apoptosis is important for the intracellular replication of the obligate intracellular pathogen Coxiella burnetii, as it allows the completion of the lengthy bacterial replication cycle. Effector proteins injected into the host cell by the C. burnetii type IVB secretion system (T4BSS) are required for the inhibition of host cell apoptosis. AnkG is one of these anti-apoptotic effector proteins. The inhibitory effect of AnkG requires its nuclear localization, which depends on p32-dependent intracellular trafficking and importin-α1-mediated nuclear entry of AnkG. Here, we compared the sequences of ankG from 37 C. burnetii isolates and classified them in three groups based on the predicted protein size. The comparison of the three different groups allowed us to identify the first 28 amino acids as essential and sufficient for the anti-apoptotic activity of AnkG. Importantly, only the full-length protein from the first group is a bona fide effector protein injected into host cells during infection and has anti-apoptotic activity. Finally, using the Galleria mellonella infection model, we observed that AnkG from the first group has the ability to attenuate pathology during in vivo infection, as it allows survival of the larvae despite bacterial replication.

Highlights

The ability to inhibit host cell apoptosis is important for the intracellular replication of the obligate intracellular pathogen Coxiella burnetii, as it allows the completion of the lengthy bacterial replication cycle
The sequences of the C. burnetii anti-apoptotic effector protein CaeA from 25 different isolates were compared, which allowed the identification of a functional domain with anti-apoptotic a ctivity[31]
We analyzed the ankG sequences from 37 C. burnetii isolates, including the 25 isolates from the CaeA study (Table 1) in order to learn more about the link between the ankG genetic background and protein function

Summary

Introduction

The ability to inhibit host cell apoptosis is important for the intracellular replication of the obligate intracellular pathogen Coxiella burnetii, as it allows the completion of the lengthy bacterial replication cycle. Only the full-length protein from the first group is a bona fide effector protein injected into host cells during infection and has anti-apoptotic activity. Cellular apoptosis is important in the defense against intracellular p athogens[13] As this is an anti-inflammatory mechanism it eliminates infected cells without inducing inflammation and tissue d estruction[14]. Antiapoptotic activity was shown for the three effector proteins AnkG, CaeA and C aeB23,26,30–33 Their exact mode(s) of interference with the host cell apoptotic machinery has not been determined. AnkG is the best-characterized C. burnetii effector protein with anti-apoptotic a ctivity[32,33] It is a 38.6 kDa protein and contains five predicted ankyrin-repeats, spanning from amino acids 15 to 80 and from 90 to 194. We aimed to increase our knowledge of the molecular requirements of AnkG activity, using dissimilarities found in the AnkG sequence from 37 C. burnetii strains

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Conclusion