Malaria parasites evade immune detection by growth and replication within erythrocytes. After erythrocyte invasion, the intracellular pathogen must increase host cell uptake of nutrients from plasma. Here, we report that the parasite-encoded RhopH complex contributes to both invasion and channel-mediated nutrient uptake. As rhoph2 and rhoph3 gene knockouts were not viable in the human P. falciparum pathogen, we used conditional knockdowns to determine that the encoded proteins are essential and to identify their stage-specific functions. We exclude presumed roles for RhopH2 and CLAG3 in erythrocyte invasion but implicate a RhopH3 contribution either through ligand-receptor interactions or subsequent parasite internalization. These proteins then traffic via an export translocon to the host membrane, where they form a nutrient channel. Knockdown of either RhopH2 or RhopH3 disrupts the entire complex, interfering with organellar targeting and subsequent trafficking. Therapies targeting this complex should attack the pathogen at two critical points in its cycle.DOI: http://dx.doi.org/10.7554/eLife.23485.001

Background: Toxin-antitoxin (TA) systems play a crucial role in bacterial survival during stress. Results: Structures of the P. vulgaris HigBA complex reveal novel structural features such as the HigB and HigA interaction and the solvent accessibility of the HigB active site. Conclusion: Antitoxin HigA interacts with toxin HigB in a novel manner. Significance: Our results emphasize that antitoxins are a structurally diverse class of proteins.

Bacteria contain multiple type II toxins that selectively degrade mRNAs bound to the ribosome to regulate translation and growth and facilitate survival during the stringent response. Ribosomedependent toxins recognize a variety of three-nucleotide codons within the aminoacyl (A) site, but how these endonucleases achieve substrate specificity remains poorly understood. Here, we identify the critical features for how the host inhibition of growth B (HigB) toxin recognizes each of the three A-site nucleotides for cleavage. X-ray crystal structures of HigB bound to two different codons on the ribosome illustrate how HigB uses a microbial RNase-like nucleotide recognition loop to recognize either cytosine or adenosine at the second A-site position. Strikingly, a single HigB residue and 16S rRNA residue C1054 form an adenosine-specific pocket at the third A-site nucleotide, in contrast to how tRNAs decode mRNA. Our results demonstrate that the most important determinant for mRNA cleavage by ribosome-dependent toxins is interaction with the third A-site nucleotide. (1,3,4). This rapid inhibitory switch suppresses metabolite consumption and temporarily halts cell growth to promote bacterial survival until nutrients are readily available. Among the prosurvival genes regulated by (p)ppGpp production are toxin-antitoxin modules, which have additional roles in antibiotic resistance and tolerance, biofilm and persister cell formation, and niche-specific colonization (5-11). The critical roles toxin-antitoxin pairs play in bacterial physiology underscore the importance of understanding their molecular targets and modes of action.There are five different classes (I to V) of toxin-antitoxin systems defined by how the antitoxin represses toxin function (1). Type II toxin-antitoxin pairs form protein-protein complexes during exponential growth that serve two purposes: inhibition of toxin activity by antitoxin binding and transcriptional autorepression to limit toxin expression (12). Antitoxins are proteolytically degraded after (p)ppGpp accumulation, leading to derepression at the toxin-antitoxin promoter (8, 12). Liberated toxin proteins inhibit the replication or translation machinery by targeting DNA gyrase, initiator tRNA fMet , glutamyl-tRNA synthetase, EF-Tu, free mRNA, ribosome-bound mRNA, and the ribosome itself (13-20).Ribosome-dependent toxins cleave mRNAs on the ribosome between the second and third nucleotides of the aminoacyl (A)-site codon (21-23). Although collectively Escherichia coli ribosome-dependent toxins target a diverse range of codons, each individual toxin appears to have a strong codon preference and cleaves at defined positions along the mRNA (24-26). RelE cleaves at UAG stop codons and the CAG sense codon (all codons denoted in the 5′-3′ direction); YoeB cleaves at codons following a translational AUG start site and at the UAA stop codon; and YafQ cleaves a single AAA sense codon (16,24,(27)(28)(29). In contrast, Proteus vulgaris host inhibition of growth B (HigB) toxin degrades multiple codons encod...