A Simple Yeast-Based Strategy to Identify Host Cellular Processes Targeted by Bacterial Effector Proteins

Bacterial effector proteins, which are delivered into the host cell via the type III secretion system, play a key role in the pathogenicity of Gram-negative bacteria by modulating various host cellular processes to the benefit of the pathogen. To identify cellular processes targeted by bacterial effectors, we developed a simple strategy that uses an array of yeast deletion strains fitted into a single 96-well plate. The array is unique in that it was optimized computationally such that despite the small number of deletion strains, it covers the majority of genes in the yeast synthetic lethal interaction network. The deletion strains in the array are screened for hypersensitivity to the expression of a bacterial effector of interest. The hypersensitive deletion strains are then analyzed for their synthetic lethal interactions to identify potential targets of the bacterial effector. We describe the identification, using this approach, of a cellular process targeted by the Xanthomonas campestris type III effector XopE2. Interestingly, we discover that XopE2 affects the yeast cell wall and the endoplasmic reticulum stress response. More generally, the use of a single 96-well plate makes the screening process accessible to any laboratory and facilitates the analysis of a large number of bacterial effectors in a short period of time. It therefore provides a promising platform for studying the functions and cellular targets of bacterial effectors and other virulence proteins

On the Performance Gap of a Generic C Optimized Assembler and Wide Vector Extensions for Masked Software with an Ascon-{\it{p}} test case

Efficient implementations of software masked designs constitute both an important goal and a significant challenge to Side Channel Analysis attack (SCA) security. In this paper we discuss the shortfall between generic C implementations and optimized (inline-) assembly versions while providing a large spectrum of efficient and generic masked implementations for any order, and demonstrate cryptographic algorithms and masking gadgets with reference to the state of the art. Our main goal is to show the prime performance gaps we can expect between different implementations and suggest how to harness the underlying hardware efficiently, a daunting task for various masking-orders or masking algorithm (multiplications, refreshing etc.). This paper focuses on implementations targeting wide vector bitsliced designs such as the ISAP algorithm. We explore concrete instances of implementations utilizing processors enabled by wide-vector capability extensions of the AMD64 Instruction Set Architecture (ISA); namely, the SSE2/3/4.1, AVX-2 and AVX-512 Streaming Single Instruction Multiple Data (SIMD) extensions. These extensions mainly enable efficient memory level parallelism and provide a gradual reduction in computation-time as a function of the level of extension and the hardware support for instruction-level parallelism. For the first time we provide a complete open-source repository of such gadgets tailored for these extensions, various gadgets types and for all orders. We evaluate the disparities between $\mathit{generic}$ high-level language masking implementations for optimized (inline-) assembly and conventional single execution path data-path architectures such as the ARM architecture. We underscore the crucial trade-off between state storage in the data-memory as compared to keeping it in the register-file (RF). This relates specifically to masked designs, and is particularly difficult to resolve because it requires inline-assembly manipulations and is not natively supported by compilers. Moreover, as the masking order ($d$) increases and the state gets larger, there must be an increase in data memory read/write accesses for state handling since the RF is simply not large enough. This requires careful optimization which depends to a considerable extent on the underlying algorithm to implement. We discuss how full utilization of SSE extensions is not always possible; i.e. when $d$ is not a power of two, and pin-point the optimal $d$ values and very sub-optimal values of $d$ which aggressively under-utilize the hardware. More generally, this paper presents several different fully generic masked implementations for any order or multiple highly optimized (inline-) assembly instances which are quite generic (for a wide spectrum of ISAs and extensions), and provide very specific implementations targeting specific extensions. The goal is to promote open-source availability, research, improvement and implementations relating to SCA security and masked designs. The building blocks and methodologies provided here are portable and can be easily adapted to other algorithms

Rationing Antiretroviral Therapy for HIV/AIDS in Africa: Choices and Consequences

The question facing African governments and societies, say Rosen and colleagues, is not whether to ration such therapy, but how to do so in a way that maximizes social welfare

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism

The Antibacterial and Anti-Eukaryotic Type VI Secretion System MIX-Effector Repertoire in <i>Vibrionaceae</i>

Vibrionaceae is a widespread family of aquatic bacteria that includes emerging pathogens and symbionts. Many Vibrionaceae harbor a type VI secretion system (T6SS), which is a secretion apparatus used to deliver toxins, termed effectors, into neighboring cells. T6SSs mediate both antibacterial and anti-eukaryotic activities. Notably, antibacterial effectors are encoded together with a gene that encodes a cognate immunity protein so as to antagonize the toxicity of the effector. The MIX (Marker for type sIX effectors) domain has been previously defined as a marker of T6SS effectors carrying polymorphic C-terminal toxins. Here, we set out to identify the Vibrionaceae MIX-effector repertoire and to analyze the various toxin domains they carry. We used a computational approach to search for the MIX-effectors in the Vibrionaceae genomes, and grouped them into clusters based on the C-terminal toxin domains. We classified MIX-effectors as either antibacterial or anti-eukaryotic, based on the presence or absence of adjacent putative immunity genes, respectively. Antibacterial MIX-effectors carrying pore-forming, phospholipase, nuclease, peptidoglycan hydrolase, and protease activities were found. Furthermore, we uncovered novel virulence MIX-effectors. These are encoded by &#8220;professional MIXologist&#8222; strains that employ a cocktail of antibacterial and anti-eukaryotic MIX-effectors. Our findings suggest that certain Vibrionaceae adapted their antibacterial T6SS to mediate interactions with eukaryotic hosts or predators

Improved Filtering Techniques for Single- and Multi-Trace Side-Channel Analysis

Side-channel analysis (SCA) attacks constantly improve and evolve. Implementations are therefore designed to withstand strong SCA adversaries. Different side channels exhibit varying statistical characteristics of the sensed or exfiltrated leakage, as well as the embedding of different countermeasures. This makes it crucial to improve and adapt pre-processing and denoising techniques, and abilities to evaluate the adversarial best-case scenario. We address two popular SCA scenarios: (1) a single-trace context, modeling an adversary that captures only one leakage trace, and (2) a multi-trace (or statistical) scenario, that models the classical SCA context. Given that horizontal attacks, localized electromagnetic attacks and remote-SCA attacks are becoming evermore powerful, both scenarios are of interest and importance. In the single-trace context, we improve on existing Singular Spectral Analysis (SSA) based techniques by utilizing spectral property variations over time that stem from the cryptographic implementation. By adapting overlapped-SSA and optimizing over the method parameters, we achieve a significantly shorter computation time, which is the main challenge of the SSA-based technique, and a higher information gain (in terms of the Signal-to-Noise Ratio (SNR)). In the multi-trace context, a profiling strategy is proposed to optimize a Band-Pass Filter (BPF) based on a low-computational cost criterion, which is shown to be efficient for unprotected and low protection level countermeasures. In addition, a slightly more computationally intensive optimized ‘shaped’ filter is presented that utilizes a frequency-domain SNR-based coefficient thresholding. Our experimental results exhibit significant improvements over a set of various implementations embedded with countermeasures in hardware and software platforms, corresponding to varying baseline SNR levels and statistical leakage characteristics

Vibrio parahaemolyticus T6SS2 effector repertoires

ABSTRACTAll strains of the marine bacterium Vibrio parahaemolyticus harbor a type VI secretion system (T6SS) named T6SS2, suggesting that this system plays an important role in the life cycle of this emerging pathogen. Although T6SS2 was recently shown to play a role in interbacterial competition, its effector repertoire remains unknown. Here, we employed proteomics to investigate the T6SS2 secretome of two V. parahaemolyticus strains, and we identified several antibacterial effectors encoded outside of the main T6SS2 gene cluster. We revealed two T6SS2-secreted proteins that are conserved in this species, indicating that they belong to the core secretome of T6SS2; other identified effectors are found only in subsets of strains, suggesting that they comprise an accessory effector arsenal of T6SS2. Remarkably, a conserved Rhs repeat-containing effector serves as a quality control checkpoint and is required for T6SS2 activity. Our results reveal effector repertoires of a conserved T6SS, including effectors that have no known activity and that have not been previously associated with T6SSs

The RIX domain defines a class of polymorphic T6SS effectors and secreted adaptors

Abstract Bacteria use the type VI secretion system (T6SS) to deliver toxic effectors into bacterial or eukaryotic cells during interbacterial competition, host colonization, or when resisting predation. Identifying effectors is a challenging task, as they lack canonical secretion signals or universally conserved domains. Here, we identify a protein domain, RIX, that defines a class of polymorphic T6SS cargo effectors. RIX is widespread in the Vibrionaceae family and is located at N-termini of proteins containing diverse antibacterial and anti-eukaryotic toxic domains. We demonstrate that RIX-containing proteins are delivered via T6SS into neighboring cells and that RIX is necessary and sufficient for T6SS-mediated secretion. In addition, RIX-containing proteins can enable the T6SS-mediated delivery of other cargo effectors by a previously undescribed mechanism. The identification of RIX-containing proteins significantly enlarges the repertoire of known T6SS effectors, especially those with anti-eukaryotic activities. Furthermore, our findings also suggest that T6SSs may play an underappreciated role in the interactions between vibrios and eukaryotes