Apicomplexan parasites comprise significant pathogens of humans, livestock, and wildlife, but also represent a diverse group of eukaryotes with interesting and unique cell biology. Study of cell biology in apicomplexan parasites is complicated by their small size, and historically this has required the application of cutting-edge microscopy techniques to investigate fundamental processes like mitosis or cell division in these organisms. Recently, a technique called expansion microscopy has been developed, which rather than increasing instrument resolution like most imaging modalities, physically expands a biological sample >4-fold. In only a few years since its development, a derivative of expansion microscopy known as ultrastructure-expansion microscopy (U-ExM) has been widely adopted and proven extremely useful for studying cell biology of Apicomplexa. Here we review the insights into apicomplexan cell biology that have been enabled through the use of U-ExM, with a specific focus on Plasmodium, Toxoplasma, and Cryptosporidium. Further, we summarise emerging expansion microscopy modifications and modalities and forecast how these may influence the field of parasite cell biology in future.

RNA-binding proteins (RBPs) govern the lifespan of nearly all transcripts and play key roles in adaptive responses in microbes. A robust approach to examine protein-RNA interactions involves irradiating cells with UV light to form covalent adducts between RBPs and their cognate RNAs. Combined with RNA or protein purification, these procedures can provide global RBP censuses or transcriptomic maps for all target sequences of a single protein in living cells. Recent development of novel methods has quickly populated the RBP landscape in microorganisms. Here, we provide an overview of prominent UV cross-linking techniques which have been applied to investigate RNA interactomes in microbes. By assessing their advantages and caveats, this technical evaluation intends to guide the selection of appropriate methods and experimental design as well as to encourage the use of complementary UV-dependent techniques to inspect RNA-binding activity.

Model organisms such as the Gram-positive bacterium Bacillus subtilis have been studied intensively for decades. However, even for such model organisms no function has been identified for about one fourth of all proteins. It has recently been realized that such understudied proteins as well as poorly studied functions set a limitation to our understanding of the requirements for cellular life, and the Understudied Proteins Initiative has been launched. Obviously, poorly studied proteins that are strongly expressed, are likely to be most important to the cell and should therefore have priority in further studies. Since the functional analysis of unknown proteins can be extremely laborious, a minimal knowledge is required prior to targeted functional studies. In this review, we discuss strategies to obtain such a minimal annotation, e.g. from global interaction, expression or localization studies. We present a set of 41 highly expressed and poorly studied proteins of B. subtilis. Several of these proteins are thought or known to bind RNA and/or the ribosome, some may may control the metabolism of B. subtilis, and another subset of particularly small proteins may act as regulatory elements to control the expression of downstream genes. Moreover, we discuss the challenges of poorly studied functions with a focus on RNA-binding proteins, amino acid transport and the control of metabolic homeostasis. The identification of the functions of the selected proteins will strongly advance our knowledge on B. subtilis, but also on other organisms since many of the proteins are conserved in many groups of bacteria.

DectiSomes are anti-infective drug-loaded liposomes targeted to pathogenic cells by pathogen receptors including the Dectins. We have previously used C-type lectin (CTL) pathogen receptors Dectin-1, Dectin-2, and DC-SIGN to target DectiSomes to the extracellular oligoglycans surrounding diverse pathogenic fungi and kill them. Dectin-3 (a.k.a. MCL, CLEC4D) is a CTL pathogen receptor whose known cognate ligands are partly distinct from other CTLs. We expressed and purified a truncated Dectin-3 polypeptide (DEC3) comprised of its carbohydrate recognition domain and stalk region. We prepared amphotericin B (AmB)-loaded pegylated liposomes (AmB-LLs) and coated them with this isoform of Dectin-3 (DEC3-AmB-LLs), and we prepared control liposomes coated with Bovine Serum Albumin (BSA-AmB-LLs). DEC3-AmB-LLs bound to the exopolysaccharide matrices of Candida albicans, Rhizopus delemar (f.k.a. R. oryzae), and Cryptococcus neoformans from one to several orders of magnitude more strongly than untargeted AmB-LLs or BSA-AmB-LLs. The data from our quantitative fluorescent binding assays were standardized using a CellProfiler program, AreaPipe, that was developed for this purpose. Consistent with enhanced binding, DEC3-AmB-LLs inhibited and/or killed C. albicans and R. delemar more efficiently than control liposomes, which significantly reduced the effective dose of AmB. In conclusion, Dectin-3 targeting has the potential to advance our goal of building pan-antifungal DectiSomes.

PlzA is a c-di-GMP-binding protein crucial for adaptation of the Lyme disease spirochete Borrelia ( Borreliella) burgdorferi during its enzootic life cycle. Unliganded apo-PlzA is important for vertebrate infection, while liganded holo-PlzA is important for tick acquisition; however, the biological function of PlzA has remained enigmatic. Here we report that PlzA has RNA chaperone activity that is inhibited by c-di-GMP binding. Holo- and apo-PlzA bind RNA and accelerate RNA annealing, while only apo-PlzA can strand displace and unwind double-stranded RNA. Guided by the crystal structure of PlzA, we identified several key aromatic amino acids protruding from the N- and C-terminal domains that are required for RNA binding and unwinding activity. Our findings illuminate c-di-GMP as a switch controlling the RNA chaperone activity of PlzA and we propose that complex RNA-mediated modulatory mechanisms allow PlzA to regulate gene expression during both the vector and host phases of the B. burgdorferi life cycle.

DNA glycosylases protect genetic fidelity during DNA replication by removing potentially mutagenic chemically damaged DNA bases. Bacterial Lhr proteins are well-characterized DNA repair helicases that are fused to additional 600-700 amino acids of unknown function, but with structural homology to SecB chaperones and AlkZ DNA glycosylases. Here we identify that E. coli Lhr is a uracil-DNA glycosylase that depends on an active site aspartic acid residue. We show that the Lhr DNA helicase activity is functionally independent of the uracil-DNA glycosylase activity, but that the helicase domains are required for fully active uracil DNA glycosylase activity. Consistent with uracil DNA glycosylase activity, deletion of lhr from the E. coli chromosome sensitized cells to oxidative stress that triggers cytosine deamination to uracil. The ability of Lhr to translocate single-stranded DNA and remove uracil bases suggests a surveillance role to seek and remove potentially mutagenic base changes during replication stress.

Figure 6

The human pathogen Staphylococcus aureus is considered mainly an extracellular, opportunistic pathogen, yet the bacterium is able to survive within and escape from host cells, including macrophages. An agr/ sae mutant of strain USA300 is unable to escape from human macrophages but can replicate and survive within macrophages. We questioned whether such “„non-toxic“” S. aureus resembles the less pathogenic coagulase-negative Staphylococcal species (CoNS) like S. carnosus, S. lugdunensis, S. capitis, S. warneri or S. pettenkoferi. We show that in contrast to the “„non-toxic“” S. aureus strains, the CoNS species are efficiently killed within 24 h post-infection in the macrophage-like THP-1 cells or in human primary macrophages. Bacterial persistence of “„non-toxic“” S . aureus or CoNS induced IL-1ß release but no cell-death. Mutations in genes coding for katalase, copprer transport or the regulatory system GraRS or SigB did not impact bacterial survival in THP-1 cells. Deletion of the superoxide dismutases sodA and sodM impaired S. aureus survival in human primary macrophages but not in THP-1 cells. However, expression of the S. aureus specific sodM in S. epidermidis was not sufficient to protect this species from being killed in THP-1 cells. Thus, at least in those cells better bacterial survival of S. aureus could not be linked to higher protection from ROS. However, “„non-toxic“” S. aureus was found to be insensitive to pH, whereas S. epidermidis was protected when phagosomal acidification was inhibited. Thus, species differences seem to be linked to different sensitivity to acidification.

Leishmania is the causative agent of the tropical neglected disease leishmaniasis and infects macrophages as its definitive host cell . In order to sustain and propagate infections, Leishmania parasites have to complete cycles of exit and re-infection. Yet, the mechanism driving the parasite spread to other cells remains unclear. Recent studies reported pro-inflammatory monocytes as replicative niche of L. major and showed prolonged expression of IL-1β at the site of infection, indicating an activation of the NLRP3 inflammasome and pointing towards pyroptosis as a possible mechanism of parasite spread. To address the species-specific inflammasome activation of human cells we characterized the BLaER1 macrophages as a model for L. major infection. We found that Leishmania can infect, activate and develop in BLaER1 macrophages similar as they can do in primary human macrophages. Harnessing the possibilities of this infection model, we first showed that BLaER1 GSDMD-/- cells, which carry a deletion of the pore-forming protein gasdermin D, are more resistant to pyroptotic cell death and, concomitantly, display a strongly delayed release of intracellular parasite. Using that knockout in a co-incubation assay in comparison with wild type BLaER1 cells, we demonstrate that impairment of the pyroptosis pathway leads to lower rates of parasite spread to new host cells, thus, implicating pyroptotic cell death as a possible exit mechanism of L. major in pro-inflammatory microenvironments.

In the last two decades, an increasing number of bacterial species have been recognized that are able to generate a phenotypically diverse population that shares an identical genotype. This ability is dependent on a complex genetic regulatory network that includes cellular and environmental signals, as well as stochastic elements. Among Bacilli, a broadly distributed family of Rap (Response-regulator aspartyl phosphate) phosphatases is known to modulate the function of the main phenotypic heterogeneity regulators by controlling their phosphorylation. Even more, their related extracellular Phr (Phosphatase regulator) peptides function as quorum sensing signals, creating a cell-cell communication network that regulates the phenotypic development of the entire population. In this review, we examine the role that the Rap phosphatases and their Phr peptides play in the regulation of B. subtilis phenotypic differentiation, and in other members of the Bacillus genus. We also highlight the contribution of these regulatory elements to the fitness of bacterial cells and mobile genetic elements, e.g. prophages and conjugative vectors.

Plasmodium is an obligate intracellular parasite that requires intense lipid synthesis for membrane biogenesis and survival. One of the principal membrane components is oleic acid, which is required to maintain the membrane’s biophysical properties and fluidity. The malaria parasite Plasmodium can modify fatty acids, and stearoyl-CoA Δ9-desaturase (Scd) is an enzyme that catalyzes the synthesis of oleic acid by desaturation of stearic acid. Scd is dispensable in P. falciparum blood stages; however, its role in mosquito and liver stages remains unknown. We show that P. berghei Scd localizes to the ER in the blood and liver stages. Disruption of Scd in the rodent malaria parasite P. berghei did not affect parasite blood stage propagation, mosquito stage development, or early liver stage development. However, when scd KO sporozoites were inoculated intravenously or by mosquito bite into mice, they failed to initiate blood-stage infection. Immunofluorescence analysis revealed that organelle biogenesis was impaired and merozoite formation was abolished, which normally initiates blood-stage infections. Genetic complementation of the KO parasites restored merozoite formation to a level similar to that of WT parasites. Mice immunized with Scd KO sporozoites confer long-lasting sterile protection against infectious sporozoite challenge. Thus, the Scd KO parasite is an appealing candidate for inducing protective preerythrocytic immunity.

Protein post-translational modifications (PTMs), such as ADP-ribosylation and phosphorylation, regulate multiple fundamental biological processes in cells. During bacterial infection, effector proteins are delivered into host cells through dedicated bacterial secretion systems and can modulate important cellular pathways by covalently modifying their host targets. These strategies enable intruding bacteria to subvert various host processes, thereby promoting their own survival and proliferation. Despite rapid expansion of our understanding of effector-mediated PTMs in host cells, analytical measurements of these molecular events still pose significant challenges in the study of host-pathogen interactions. Nevertheless, with major technical breakthroughs in the last two decades, mass spectrometry (MS) has evolved to be a valuable tool for detecting protein PTMs and mapping modification sites. Additionally, large-scale PTM profiling, facilitated by different enrichment strategies prior to MS analysis, allows high-throughput screening of host enzymatic substrates of bacterial effectors. In this review, we summarize the advances in the studies of two representative PTMs (i.e., ADP-ribosylation and phosphorylation) catalyzed by bacterial effectors during infection. Importantly, we will discuss the ever increasing role of MS in understanding these molecular events and how the latest MS-based tools can aid in future studies of this booming area of pathogenic bacteria-host interactions.

Super-resolution fluorescence microscopy techniques developed over the past two decades have pushed the resolution limit for fluorescently labeled molecules into the nanometer range. These techniques have the potential to study bacterial macromolecular complexes such as secretion systems with single-molecule resolution on a millisecond time scale. Here we review recent applications of super-resolution fluorescence microscopy in molecular bacteriology with a focus on bacterial secretion systems. We also describe MINFLUX fluorescence nanoscopy, a relatively new technique that promises to one day produce molecular movies of bacterial molecular machines in action.

YbeX of Escherichia coli, a member of the CorC protein family, is encoded in the same operon with ribosome-associated proteins YbeY and YbeZ. Here, we report the involvement of YbeX in ribosomal metabolism. The ΔybeX cells accumulate distinct 16S rRNA degradation intermediates in the 30S particles and the 70S ribosomes. E. coli lacking ybeX has a lengthened lag phase upon outgrowth from the stationary phase. This growth phenotype is heterogeneous at the individual cell level and especially prominent under low extracellular magnesium levels. The ΔybeX strain is sensitive to elevated growth temperatures and to several ribosome-targeting antibiotics that have in common the ability to induce the cold shock response in E. coli. Although generally milder, the phenotypes of the ΔybeX mutant overlap with those caused by ybeY deletion. A genetic screen revealed partial compensation of the ΔybeX growth phenotype by the overexpression of YbeY. These findings indicate an interconnectedness amongst the ybeZYX operon genes, highlighting their roles in ribosomal assembly and/or degradation.

The enteropathogen Salmonella enterica subsp. enterica sv. Typhimurium str. LT2 (hereafter S. Typhimurium) utilizes a cluster of genes encoded within the pathogenicity island 2 (SPI-2) of its genome to proliferate inside macrophages. The expression of SPI-2 is controlled by a complex network of transcriptional regulators and environmental cues, which now include a recently characterized DNA-binding protein named PagR. Growth of S. Typhimurium in low phosphate low magnesium medium mimics conditions inside macrophages. Under such conditions, PagR ensures SPI-2 induction by upregulating the transcription of slyA, a known activator of SPI-2. Here we report that PagR represses the expression of a divergently transcribed polycistronic operon that encodes the two subunits of transketolase TktC ( i.e., tktD, tktE) of this bacterium. Transketolases contribute to the non-redox rearrangements of phosphorylated sugars of the pentose phosphate pathway, which provide building blocks for amino acids, nucleotides, cofactors, etc. We also demonstrate that PagR represses the expression of its own gene and define two PagR binding sites between stm2344 and pagR.

Evolutionary studies often identify genes that are shared between different organisms and the phrase Lateral or Horizontal Gene Transfer is often used in this context. However, they rarely provide any mechanistic information concerning how these gene transfers might have occurred. With the astonishing increase in the number of sequences in the public databases over the past two or three decades, identical antibiotic resistance genes have been identified in many different sequence contexts. One explanation for this would be that genes are initially transmitted by transposons which have subsequently decayed and can no longer be detected. Here, we provide an overview of a protein, IEE (Insertion Sequence Excision Enhancer) observed to facilitate high frequency excision of IS 629 from clinically important Escherichia coli O157:H7 and subsequently shown to affect a large class of bacterial insertion sequences which all transpose by using the copy-out-paste-in transposition mechanism. Excision depends on both IEE and transposase indicating association with the transposition process itself. We review genetic and biochemical data and propose that IEE immobilizes genes carried by compound transposons by removing the flanking IS copies. The biochemical activities of IEE as a primase with the capacity to recognize DNA microhomologies and the observation that its effect appears restricted to IS families which use copy-out-paste-in transposition, suggests IS deletion occurs by abortive transposition involving strand switching during the copy-out step. This reinforces the proposal made for understanding loss of IS Apl1 flanking mcr-1 in the compound transposon Tn 6330 which we illustrate with a detailed model.

Translation of messenger RNA (mRNA) in bacteria occurs in the steps of initiation, elongation, termination and ribosome recycling. The initiation step comprises multiple stages and uses a special transfer RNA (tRNA) called initiator tRNA (i-tRNA), which is first aminoacylated and then formylated using methionine and N 10-formyl-tetrahydrofolate (N 10-fTHF), respectively. Both methionine and N 10-fTHF are produced via one-carbon metabolism, linking translation initiation with active cellular metabolism. The fidelity of i-tRNA binding to the ribosomal peptidyl-site (P-site) is attributed to the structural features in its acceptor stem, and the highly conserved three consecutive G-C base pairs (3GC pairs) in the anticodon stem. While the acceptor stem region is important in formylation of the amino acid attached to i-tRNA and its initial binding to the P-site, the 3GC pairs are crucial in transiting i-tRNA through various stages of initiation. We utilized the feature of 3GC pairs to investigate the nuanced layers of scrutiny that ensure fidelity of translation initiation through i-tRNA abundance and its interactions with the components of the translation apparatus. We discuss the importance of i-tRNA in the final stages of ribosome maturation, as also the roles of the Shine-Dalgarno sequence, ribosome heterogeneity, initiation factors, ribosome recycling factor and coevolution of the translation apparatus in orchestrating a delicate balance between the fidelity of initiation and/or its leakiness to generate proteome plasticity in cells to confer growth fitness advantages in response to the dynamic nutritional states.