Adaptive evolution of the Streptococcus pyogenes regulatory aldolase LacD.1

In the human-pathogenic bacterium Streptococcus pyogenes, the tagatose bisphosphate aldolase LacD.1 likely originated through a gene duplication event and was adapted to a role as a metabolic sensor for regulation of virulence gene transcription. Although LacD.1 retains enzymatic activity, its ancestral metabolic function resides in the LacD.2 aldolase, which is required for the catabolism of galactose. In this study, we compared these paralogous proteins to identify characteristics correlated with divergence and novel function. Surprisingly, despite the fact that these proteins have identical active sites and 82% similarity in amino acid sequence, LacD.1 was less efficient at cleaving both fructose and tagatose bisphosphates. Analysis of kinetic properties revealed that LacD.1's adaptation was associated with a decrease in k(cat) and an increase in K(m). Construction and analysis of enzyme chimeras indicated that non-active-site residues previously associated with the variable activities of human aldolase isoenzymes modulated LacD.1's affinity for substrate. Mutant LacD.1 proteins engineered to have LacD.2-like levels of enzymatic efficiency lost the ability to function as regulators, suggesting that an alteration in efficiency was required for adaptation. In competition under growth conditions that mimic a deep-tissue environment, LacD.1 conferred a significant gain in fitness that was associated with its regulatory activity. Taken together, these data suggest that LacD.1's adaptation represents a form of neofunctionalization in which duplication facilitated the gain of regulatory function important for growth in tissue and pathogenesis

Citrulline protects Streptococcus pyogenes from acid stress using the arginine deiminase pathway and the F1Fo-ATPase

A common stress encountered by both pathogenic and environmental bacteria is exposure to a low-pH environment, which can inhibit cell growth and lead to cell death. One major defense mechanism against this stress is the arginine deiminase (ADI) pathway, which catabolizes arginine to generate two ammonia molecules and one molecule of ATP. While this pathway typically relies on the utilization of arginine, citrulline has also been shown to enter into the pathway and contribute to protection against acid stress. In the pathogenic bacterium Streptococcus pyogenes, the utilization of citrulline has been demonstrated to contribute to pathogenesis in a murine model of soft tissue infection, although the mechanism underlying its role in infection is unknown. To gain insight into this question, we analyzed a panel of mutants defective in different steps in the ADI pathway to dissect how arginine and citrulline protect S. pyogenes in a low-pH environment. While protection provided by arginine utilization occurred through the buffering of the extracellular environment, citrulline catabolism protection was pH independent, requiring the generation of ATP via the ADI pathway and a functional F(1)F(o)-ATP synthase. This work demonstrates that arginine and citrulline catabolism protect against acid stress through distinct mechanisms and have unique contributions to virulence during an infection. IMPORTANCE An important aspect of bacterial pathogenesis is the utilization of host-derived nutrients during an infection for growth and virulence. Previously published work from our lab identified a unique role for citrulline catabolism in Streptococcus pyogenes during a soft tissue infection. The present article probes the role of citrulline utilization during this infection and its contribution to protection against acid stress. This work reveals a unique and concerted action between the catabolism of citrulline and the F(1)F(o)-ATPase that function together to provide protection for bacteria in a low-pH environment. Dissection of these collaborative pathways highlights the complexity of bacterial infections and the contribution of atypical nutrients, such as citrulline, to pathogenesis

SpxA1 and SpxA2 act coordinately to fine-tune stress responses and virulence in Streptococcus pyogenes

SpxA is a unique transcriptional regulator highly conserved among members of the phylum Firmicutes that binds RNA polymerase and can act as an antiactivator. Why some Firmicutes members have two highly similar SpxA paralogs is not understood. Here, we show that the SpxA paralogs of the pathogen Streptococcus pyogenes, SpxA1 and SpxA2, act coordinately to regulate virulence by fine-tuning toxin expression and stress resistance. Construction and analysis of mutants revealed that SpxA1− mutants were defective for growth under aerobic conditions, while SpxA2− mutants had severely attenuated responses to multiple stresses, including thermal and oxidative stresses. SpxA1− mutants had enhanced resistance to the cationic antimicrobial molecule polymyxin B, while SpxA2− mutants were more sensitive. In a murine model of soft tissue infection, a SpxA1− mutant was highly attenuated. In contrast, the highly stress-sensitive SpxA2− mutant was hypervirulent, exhibiting more extensive tissue damage and a greater bacterial burden than the wild-type strain. SpxA1− attenuation was associated with reduced expression of several toxins, including the SpeB cysteine protease. In contrast, SpxA2− hypervirulence correlated with toxin overexpression and could be suppressed to wild-type levels by deletion of speB. These data show that SpxA1 and SpxA2 have opposing roles in virulence and stress resistance, suggesting that they act coordinately to fine-tune toxin expression in response to stress. SpxA2− hypervirulence also shows that stress resistance is not always essential for S. pyogenes pathogenesis in soft tissue

Host and bacterial proteases influence biofilm formation and virulence in a murine model of enterococcal catheter-associated urinary tract infection

Urinary tract infections: targeting enzymes might help Identifying bacterial and host enzymes that support biofilm formation may help prevent urinary tract infections caused by catheters. Enterococcus faecalis bacteria is a leading cause of catheter-associated urinary tract infections, the most common type of hospital-acquired infections. Michael Caparon and colleagues at Washington University School of Medicine in Missouri, USA, studied these infections in mice. They examined the effects of two protein-degrading enzymes, both from the bacterium and one can be activated by urine trypsin-like protease from the animals. Mutations that impaired either one of the enzymes had no effect on the infection, but when both the bacterial enzymes were impaired by mutation the formation of biofilms was significantly reduced. Treating the mice with chemicals that inhibited both bacterial and host enzymes dramatically reduced catheter-induced inflammation and related problems. This suggests drugs targeting these enzymes could be useful in clinical care

The "Software Factory" reconsidered: An Approach to the Strategic Management of Engineering

"May 1987.

Services and the Business Models of Product Firms: An Empirical Analysis of the Software Industry

Some product firms increasingly rely on service revenues as part of their business models. One possible explanation is that they turn to services to generate additional profits when their product industries mature and product revenues and profits decline. We explore this assumption by examining the role of services in the financial performance of firms in the prepackaged software products industry (Standard Industrial Classification code 7372) from 1990 to 2006. We find a convex, nonlinear relationship between a product firm's fraction of total sales coming from services and its overall operating margins. As expected, firms with a very high level of product sales are most profitable, and rising services are associated with declining profitability. We find, however, that additional services start to have a positive marginal effect on the firm's overall profits when services reach a majority of a product firm's sales. We show that traditional industry maturity arguments cannot fully explain our data. It is likely that changes in both strategy and the business environment lead product firms to place more emphasis on services

Examining the Validity of Using a Gaussian Schell-Model Source To Model the Scattering of A Fully Coherent Gaussian Beam From A Rough Impedance Surface

Military applications that use adaptive optics (AO) often require a point source beacon at the target to measure and correct for wavefront aberrations introduced by atmospheric turbulence. However, turbulence prevents the formation of such a point beacon. The extended beacons that are created instead have finite spatial extents and exhibit varying degrees of spatial coherence. Modeling these extended beacons using a Gaussian Schell-model (GSM) form for the autocorrelation function would be a convenient approach due to the analytical tractability of Gaussian functions. We examine the validity of using such a model by evaluating the field scattered from a rough impedance surface using a full-wave computational technique called the method of moments (MoM). The MoM improves the fidelity of the analysis since it captures all the physics of the laser-target interaction, such as masking, shadowing, multiple reflections, etc. Two rough-surface targets with different roughness statistics are analyzed. The simulation results are verified with experimental bidirectional reflectance distribution function measurements. It is seen that for rough surfaces, in general, the scattered-field autocorrelation function is not of a GSM form. However, under certain conditions, modeling an extended beacon as a GSM source is legitimate. This analysis will aid in understanding the behavior of extended beacons and how they affect the overall performance of an AO system

Ejection-accretion connection in NLS1 AGN 1H 1934-063

Accretion and ejection of matter in active galactic nuclei (AGN) are tightly connected phenomena and represent fundamental mechanisms regulating the growth of the central supermassive black hole and the evolution of the host galaxy. However, the exact physical processes involved are not yet fully understood. We present a high-resolution spectral analysis of a simultaneous \xmm\ and \nustar\ observation of the narrow line Seyfert 1 (NLS1) AGN 1H 1934-063, during which the X-ray flux dropped by a factor of $\sim6$ and subsequently recovered within 140 kiloseconds. By means of the time-resolved and flux-resolved X-ray spectroscopy, we discover a potentially variable warm absorber and a relatively stable ultra-fast outflow (UFO, $v_\mathrm{UFO}\sim-0.075\,c$) with a mild ionization state ($\log(\xi/\mathrm{erg\,cm\,s^{-1})}\sim1.6$). The detected emission lines (especially a strong and broad feature around 1\,keV) are of unknown origin and cannot be explained with emission from plasmas in photo- or collisional-ionization equilibrium. Such emission lines could be well described by a strongly blueshifted ($z\sim-0.3$) secondary reflection off the base of the equatorial outflows, which may reveal the link between the reprocessing of the inner accretion flow photons and the ejection. However, this scenario although being very promising is only tentative and will be tested with future observations.Comment: 15 pages, 17 figures, 1 table, accepted for publication in MNRA

Combinatorial small-molecule therapy prevents uropathogenic Escherichia coli catheter-associated urinary tract infections in mice

Catheter-associated urinary tract infections (CAUTIs) constitute the majority of nosocomial urinary tract infections (UTIs) and pose significant clinical challenges. These infections are polymicrobial in nature and are often associated with multidrug-resistant pathogens, including uropathogenic Escherichia coli (UPEC). Urinary catheterization elicits major histological and immunological alterations in the bladder that can favor microbial colonization and dissemination in the urinary tract. We report that these biological perturbations impact UPEC pathogenesis and that bacterial reservoirs established during a previous UPEC infection, in which bacteriuria had resolved, can serve as a nidus for subsequent urinary catheter colonization. Mannosides, small molecule inhibitors of the type 1 pilus adhesin, FimH, provided significant protection against UPEC CAUTI by preventing bacterial invasion and shifting the UPEC niche primarily to the extracellular milieu and on the foreign body. By doing so, mannosides potentiated the action of trimethoprim-sulfamethoxazole in the prevention and treatment of CAUTI. In this study, we provide novel insights into UPEC pathogenesis in the context of urinary catheterization, and demonstrate the efficacy of novel therapies that target critical mechanisms for this infection. Thus, we establish a proof-of-principle for the development of mannosides to prevent and eventually treat these infections in the face of rising antibiotic-resistant uropathogens